Methods Of Treatment With Angiopoietin-2 Antibodies

ABSTRACT

The disclosure is directed to methods and uses of antibodies or antigen-binding fragments thereof against Angiopoietin-2 (Ang-2). Specifically, the disclosure is direct to the use of anti-Ang2 antibodies or antigen-binding fragments thereof for treating ischemia. The methods disclosed are useful for reducing microvascular permeability, increasing microvascular perfusion, reducing inflammation in a tissue, and treating or ameliorating diseases associated with ischemia and/or reperfusion injury. The disclosed methods are also useful for protecting solid organ transplant tissue and treating or preventing chronic tissue transplant rejection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/608,314, filed Mar. 8, 2012 and U.S. Provisional Application No.61/638,303, filed Apr. 25, 2012 which is incorporated by reference intheir entirety.

SEQUENCE LISTING

The content of the electronically submitted sequence listing in ASCIItext file (Name: “SEQIDListing.ascii.txt”; Size: 14,280 bytes; and Dateof Creation: Apr. 24, 2012) filed with the application is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to methods and uses for antibodies orantigen-binding fragments thereof against Angiopoietin-2 (Ang-2).Aspects of the invention relate to use of anti-Ang2 antibodies orantigen-binding fragments thereof for treating ischemia. The methodsdisclosed herein are useful for reducing microvascular permeability in atissue; increasing microvascular perfusion in a tissue; reducinginflammation in a tissue; and treating or ameliorating diseasesassociated with ischemia and/or reperfusion injury. The disclosedmethods are also useful for protecting solid organ transplant tissue andtreating or preventing chronic tissue transplant rejection, e.g.,treating or preventing ischemic reperfusion injury and microvasculardysfunction in cardiac allografts.

2. Background

Angiopoietins were discovered as ligands for Ties, a family of tyrosinekinases that is selectively expressed within the vascular endothelium(Yancopoulos et al., Nature 407:242-48 (2000)). There are now at leastfour members of the angiopoietin family: Angiopoietin-1, -2, -3 and -4.Ang-3 and Ang-4 represent widely diverged counterparts of the same genelocus in mouse and man (Kim et al., FEBS Let, 443:353-56 (1999); Kim etal., J Biol Chem 274:26523-28 (1999)). Ang-1 and Ang-2 are endothelialcell (EC)-specific growth factors, acting through the same tyrosinekinase receptor, Tie2 (1-3). Ang-1 and Ang-2 were originally identifiedin tissue culture experiments as an agonist and an antagonist,respectively (Davis et al., Cell 87:1161-69 (1996); Maisonpierre et al.,Science 277:55-60 (1997)). All of the known angiopoietins bind primarilyto Tie2, and both Ang-1 and -2 bind to Tie2 with an affinity of 3 nM(Kd) (Maisonpierre et al., Science 277:55-60 (1997)). Ang-1 was shown tosupport EC survival and to promote endothelium integrity (Davis et al.,Cell 87:1161-69 (1996); Kwak et al., FEBS Lett 448:249-53 (1999); Suriet al., Science 282:468-71 (1998); Thurston et al., Science 286: 2511-14(1999); and Thurston et al., Nat. Med. 6:460-63 (2000)), whereas Ang-2had the opposite effect and promoted blood vessel destabilization andregression in the absence of the survival factors VEGF or basicfibroblast growth factor (Maisonpierre et al., Science 277:55-60(1997)).

Angiopoietins-1 and -2 (Ang1, 2) are endothelial cell specific growthfactors with context-dependent effects on their receptor, Tie2,including Ang-1 stabilization and Ang-2 destabilization of endothelialcell-cell junctions in mature vessels. Ang1 is constitutively secretedby pericytes and smooth muscle cells (SMCs) that are associated withblood vessels, and Ang1 has an indispensable role in embryonic vasculardevelopment and vessel maturation. Knocking Ang1 out, evenheart-specifically, is fatal (4-6). Besides its role in embryogenesis,Ang1 also stabilizes immature and leaky vessels by promotinginteractions between the endothelial cells (ECs) and perivascularstructures and by regulating the endothelial cytoskeleton. Ang1 reducesvascular leakage and infarct size in ischemic murine brain model (7). Acartilage oligomeric matrix protein—Ang1 fusion, a soluble and stablevariant of native Ang1 engineered to have higher activity, reducesendotoxemia-induced acute kidney injury, as well as experimentalIRI-induced acute kidney injury (8), (9), (10).

In contrast, Ang-2 selectively competes with Ang-1 for binding to theTie2 receptor and has a context dependent effect on the receptor (3,11-13). Ang-2 is an agonist or antagonist depending on the context. Ang2is stored together with von Willebrand factor, P-selectin, andendothelin-1 (ET-1) in Weibel-Palade bodies in the EC, and is releasedin response to homeostatic reactions such as inflammation andcoagulation (14). Ang-2 is released in response to stress, such asinflammatory stimuli. However, Ang-2 has the ability to enhanceneovascularization and to promote EC survival or increase vascularpermeability (15-17). Furthermore, Ang-2 production is increased inhypoxic EC and in early phases of angiogenesis, it is involved in vesseldestabilization. Induction of Ang-2 in the absence of vascularendothelial growth factor (VEGF) leads to endothelial apoptosis, whilein the presence of VEGF, it promotes angiogenesis. The duration of Ang-2exposure appears to have differing effects on vascular integrity inhindlimb ischemia models. For example, transient exposure appears todemonstrate a neovascularization role for Ang-2 (18) and extendedexposure to Ang-2 appears to increase inflammation and vascular leakage(19). Ang-2 mRNA expression and protein levels are increased afterexperimental murine myocardial ischemia-reperfusion injury (IRI) (20).The specific role of Ang1/Ang2 axis in ischemia-reperfusion injury isnot yet fully elucidated.

Hypothermic preservation is the gold standard of preoperative allografttreatment in solid organ transplantation. The restoration of blood flowfollowing hypothermic preservation can result in profound IRI andmicrovascular dysfunction as well as the activation of innate immuneresponses, which is linked to the development of chronic rejection insolid organ transplantation. IRI can initiate pathological immuneresponses through the hypoxia response and release of endogenous dangermolecules such as biglycan, hyaluronan and high mobility group box-1(HMGB1) protein (21-23). These danger/damage-associated molecularpatterns (DAMPs) ligate to Toll-like receptors (TLRs) and form a linkbetween the innate immunity and the activation of the adaptive immunitythat can ultimately be detrimental for the cardiac allograft and thepatient (24, 25). Current organ preservation solutions fail to eliminateIRI completely, hence, finding additional methods of organ protection isneeded.

While reperfusion is favorable in terms of myocardial salvage, it canresult in additional cardiac damage, e.g., IRI. IRI has been associatedwith worsening or expansion of the prior ischemic damage resulting inmicrovascular dysfunction arising from endothelial cell damage,stunning, reperfusion arrhythmias, and myocyte death; a contributor tothese effects is free radical generation. Thus, reperfusion injuryremains a major therapeutic challenge. IRI is an inevitable factor incardiac surgery and clinical transplantation, and new ways of itsprimary prevention and cardiac preservation are actively beinginvestigated. IRI is associated with disruption of vascular endothelialintegrity, EC damage and subsequent pathological remodeling leading tochronic rejection.

Thus, there is a need to develop methods for treating ischemic diseases,e.g., ischemia-reperfusion injury; reducing microvascular permeabilityin a tissue; increasing microvascular perfusion in a tissue; reducinginflammation in a tissue; and treating or ameliorating diseasesassociated with ischemia and/or reperfusion. Furthermore, there is aneed to develop methods to protect solid organ transplant tissue andmethods of treating or preventing chronic tissue transplant rejection.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

BRIEF SUMMARY OF THE INVENTION

In certain aspects, the invention is directed to a method for reducingmicrovascular permeability in a tissue, comprising administering atherapeutically effective amount of an anti-Ang2 antibody orantigen-binding fragment thereof to a tissue in need thereof.

In another aspect, the invention is directed to a method for increasingmicrovascular perfusion in a tissue, comprising administering atherapeutically effective amount of an anti-Ang2 antibody orantigen-binding fragment thereof to a tissue in need thereof.

In another aspect, the invention is directed to a method for reducinginflammation in a tissue, comprising administering a therapeuticallyeffective amount of an anti-Ang2 antibody or antigen-binding fragmentthereof to a tissue in need thereof.

In another aspect, the invention is directed to a method for protectinga solid organ transplant tissue, comprising administering an effectiveamount of an anti-Ang2 antibody or antigen-binding fragment thereof toan allograft.

In another aspect, the invention is directed to a method for preventingchronic rejection, comprising administering a therapeutically effectiveamount of an anti-Ang2 antibody or antigen-binding fragment thereof toan allograft.

In another aspect, the invention is directed to a method for treatingchronic allograft vasculopathy (CAV) in a subject comprisingadministering a therapeutically effective amount of an anti-Ang2antibody or antigen-binding fragment thereof to an allograft.

In certain embodiments, the tissue is selected from the group consistingof heart, kidney, brain, smooth muscle, and intestine tissue. In someembodiments, the tissue is an allograft. In some embodiments, theallograft is a cardiac allograft. In certain embodiments, the tissue orallograft is at risk for or has suffered from ischemic reperfusioninjury. In certain embodiments, the allograft is perfused with theanti-Ang2 antibody or antigen-binding fragment thereof.

In another aspect, the invention is directed to a method for treating orameliorating myocardial ischemia, comprising administering atherapeutically effective amount of an anti-Ang2 antibody orantigen-binding fragment thereof to a subject in need thereof.

In another aspect, the invention is directed to a method for treating anIRI-induced inflammatory response in a subject comprising administeringa therapeutically effective amount of an anti-Ang2 antibody orantigen-binding fragment thereof to the subject in need thereof.

In certain embodiments, administration of the anti-Ang2 antibody orantigen-binding fragment thereof reduces the immunoreactivity of VCAM-1.

In certain embodiments, the anti-Ang2 antibody or antigen-bindingfragment thereof neutralizes Ang-2.

In certain embodiments, the anti-Ang2 antibody or antigen-bindingfragment thereof preferentially binds Ang-2 over Ang-1.

In certain embodiments, the anti-Ang2 antibody or antigen-bindingfragment thereof is an antibody that binds to and neutralizes Ang-2, butdoes not bind to Ang-1.

In certain embodiments, the anti-Ang2 antibody or antigen-bindingfragment thereof comprises a variable light chain (VL) comprising CDRs1-3 of SEQ ID NO: 3, 4, 5, 6, or 8 and a variable heavy chain (VH)comprising CDRs 1-3 of SEQ ID NO: 7. In some embodiments, the VLcomprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 8 and theVH comprises the amino acid sequence of SEQ ID NO: 7. In someembodiments, the anti-Ang2 antibody or antigen-binding fragment thereofis Ang-2 antibody binds to the same epitope as MEDI1/5, MEDI2/5,MEDI3/5, MEDI4/5, or MEDI6/5.

In certain embodiments, the anti-Ang2 antibody or antigen-bindingfragment thereof is Ang-2 antibody competitively inhibits binding ofMEDI1/5, MEDI2/5, MEDI3/5, MEDI4/5, or MEDI6/5 to Ang-2.

In certain embodiments, the anti-Ang2 antibody or antigen-bindingfragment thereof is Ang-2 antibody MEDI1/5 or a derivative thereof.

In certain embodiments, the anti-Ang2 antibody or antigen-bindingfragment thereof is administered in a single dose. In some embodiments,the anti-Ang2 antibody or antigen-binding fragment thereof isadministered preoperatively. In some embodiments, the anti-Ang2 antibodyor antigen-binding fragment thereof is administered to the allograftdonor, to the allograft or to the allograft recipient.

In certain embodiments, the anti-Ang2 antibody or antigen-bindingfragment thereof reduces expression of VEGF, TGFβ, or both.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows hypoxia induces Ang2 deposition in endothelial cell-celljunctions. Human dermal blood microvascular cells (BECs) transfectedwith Tie2-GFP retrovirus were subjected to normoxia or hypoxia (1% O2) 5hours after plating for 16 hours in the presence of control-IgG oranti-Ang2 antibody (MEDI1/5) (2 μg/ml). Immunofluorecent staining showsthe localization of Tie2 and Ang2; DAPI indicates nuclei. (Scale bars=20μm).

FIG. 2 shows intracoronary perfusion with anti-Ang2 antibody (MEDI1/5),and not control-IgG, stabilizes rat cardiac allograft endothelium duringpreservation. (A) Shows the incidence of EC gaps (%) in microvascularendothelial cell (EC)-EC junctions determined by transmission electronmicroscopy in non-transplanted rat cardiac allografts. (B) Shows theincidence of EC blebbing (%). (C-D) Show representative images of EC-ECgaps and blebbing in transmission electron microscopy: red arrowsindicate gaps in EC-EC junctions, black arrows indicate EC blebbing. n=5per group. The Mann-Whitney U test was used to compare anti-Ang2 and IgGtreatment. Data are given by box plots showing the upper extreme(excluding outliers), upper quartile, median, lower quartile, and lowerextreme (excluding outliers) (A and B). Rbc indicates red blood cell.Magnification 25000×; scale bars=1 μm (C and D). *P<0.05.

FIG. 3 shows that the anti-Ang2 antibody (MEDI1/5) reduces vascularendothelium permeability and increases the number of perfusedcapillaries after cardiac allograft IRI. (A) Shows vascular permeabilityresults measured by quantifying Evans Blue absorbance from themyocardium at 30 minutes after reperfusion. Evans Blue from themyocardium treated with anti-Ang2 antibody is compared to control IgG at30 min after reperfusion (p<0.05). (B) Shows capillary perfusion resultsmeasured by quantifying tomato lectin positive capillaries from cardiaccross sections from at least 10 random immunofluorescent images. Themean density of the capillaries was measured by quantifying Lycopersiconesculentum (tomato) lectin positive vessels in histological sections,anti-Ang2 antibody treatment compared to control allografts, at 30 min.**p<0.01, n=8 per group. (C-D) Show examples of control IgG andanti-Ang2 antibody tomato lectin positive capillaries from cardiac crosssections. Magnification 100×, inset 400× (Scale bar represents 20 μm).(E) Shows the tissue perfusion in the apical myocardium of cardiacallografts determined by Doppler velocimeter during the first 10 minafter reperfusion. n=8 per group; repeated measurements ANOVA (E).

FIG. 4 shows intracoronary perfusion with recombinant Ang2 has no effecton microvascular dysfunction 30 min after reperfusion, but increasesmyocardial injury 6 h after reperfusion. (A) Shows vascular permeabilitymeasured by quantifying myocardial absorbance of extravasated Evans Bluedye with spectrofotometry at 610 nm 30 min after reperfusion. (B-D) Showmyocardial capillary perfusion measured by quantifying the density ofendothelium-binding FITC-conjugated Lycopersicon esculentum (tomato)lectin (green). (E) Shows the serum levels of cardiomyocyte specifictroponin T (TnT). n=5 to 6 per group. The Mann-Whitney U test was usedto compare recombinant (rAng2) and PBS treatment. Magnification 100×,inset 400× (Scale bars=20 μm). (F-J) Shows ex vivo intracoronaryperfusion with exogenous recombinant Ang2 worsens allograftinflammation. The numbers of allograft-infiltrating ED1+ macrophages,MPO+ neutrophils, CD4+ T cells, CD8+ T cells, and OX62+ dendritic cells6 h after reperfusion (n=6 per group). Data are given by box plotsshowing the upper extreme (excluding outliers), upper quartile, median,lower quartile, and lower extreme (excluding outliers). *P<0.05 usingthe Mann-Whitney U test (F-J).

FIG. 5 shows intracoronary perfusion with anti-Ang2 reduced theimmunoreactivity of Ang2 6 h after reperfusion. (A) Shows the expressionof endothelial Ang2 measured by quantifying the density of Ang2/ratendothelial cell antigen (RECA-1) double positive vessels 6 h aftertransplantation (n=6 per group). (B) Shows myocardial Ang2 expressionvisualized by immunofluorescence staining for Ang2 and RECA-1. TheMann-Whitney U test was used to compare anti-Ang2 and IgG treatment.Data are given by box plots showing the upper extreme (excludingoutliers), upper quartile, median, lower quartile, and lower extreme(excluding outliers). **P<0.01 (Scale bars=20 μm).

FIG. 6 shows that the anti-Ang2 antibody (MEDI1/5) decreases earlymyocardial damage, inflammatory cell influx, adhesion moleculeexpression and early innate immune activation. The allografts wereintracoronarily perfused with anti-Ang2 antibody or control IgG andsubjected to a 4-h hypothermic preservation, transplanted and recoveredat 6 h after reperfusion. (A) Shows serum levels of cardiomyocytespecific troponin T (TnT). n=6-9 per group. (B-F) Show quantification ofintragraft ED1+ macrophages, MPO+ neutrophils, CD4+ and CD8+ lymphocytesand OX62+DCs, respectively. n=8-9 per group. (G and J) Showsquantification of VCAM-1+ and ICAM-1+ vessels, respectively, and (H-Iand K-L) show representative histological images of VCAM-1+ and ICAM-1+vessels from control IgG and anti-Ang2 antibody treated groups,respectively. n=8-9 per group. The Mann-Whitney U test was used tocompare anti-Ang2 and IgG treatment. *p<0.05; **p<0.01; ***p<0.001.(Scale bar represents 20 μm).

FIG. 7 shows that anti-Ang2 antibody (MEDI1/5) reduces cardiac fibrosisand cardiac allograft vasculopathy 8 weeks after transplantation. Theallografts were intracoronarily perfused with the anti-Ang2 antibody orcontrol IgG and subjected to a 2-h hypothermic preservation,transplanted and recovered at 8 weeks. (A) Shows the results forquantification of cardiac fibrosis assessed with Masson's trichromestaining for control IgG and anti-Ang2 antibody treated groups. Computerassisted analysis was performed to measure the area stained blue forfibrosis from at least 10 images. (B-C) Show examples of cardiac crosssections with Masson's trichrome staining for control IgG and anti-Ang2antibody, respectively. (D) Shows the results for quantification of thedegree of occlusion for control IgG and anti-Ang2 antibody treatedgroups. Data are indicated as mean±SEM, analyzed with Mann-WhitneyU-test; n=8 per group. *p<0.05; **p<0.01. (E-F) Show examples ofHematoxylin-eosin and Resorcin-Fuchsin stained histological sections fordetection of internal elastic lamina as black and visualization of theneointima and occlusion of arteries in control IgG and anti-Ang2antibody treated groups, respectively. The Mann-Whitney U test was usedto compare anti-Ang2 and IgG treatment. (Scale bar represents 20 μm).(G) Shows the results for quantification of the number of RECA-1+stained vessels for control IgG and anti-Ang2 antibody treated groups.(H-I) Shows examples of RECA-1+ stained vessels for control IgG andanti-Ang2 antibody treated groups. n=8/group. Magnification 100×, inset400×.

FIG. 8 shows Ang2 and Ang1 levels in serum from human cardiac allograftdonors and recipients. (A-B) Shows Ang2 and Ang1 levels in patient serumsamples from cardiac allograft donors (n=11) determined by enzyme-linkedimmunosorbent assay (ELISA) and compared to healthy controls (n=24).(C-D) Shows Ang2 and Ang1 levels in patient serum samples from cardiacallograft recipients (n=11) 1, 6, 12, and 24 h after transplantationdetermined by ELISA and compared to healthy controls (n=24). (E) ShowsAng2 levels in rat serum samples taken from cardiac allograft recipientsand analyzed by ELISA preoperatively, 1, 6, 24 h after transplantation(n=8). The Mann-Whitney U test was used to compare the control group andthe donor group (A and B). The Kruskall-Wallis test with Dunn correctionwas used to compare the control group with the recipient groups (C andD). Data are given by box plots showing the upper extreme (excludingoutliers), upper quartile, median, lower quartile, and lower extreme(excluding outliers) (A-D): *P<0.05, ***P<0.001. Repeated measurementsANOVA was used to compare the sample groups (E): *P=0.0001, **P=1.48E-5,***P=1.85E-7.

FIG. 9 shows ex vivo intracoronary treatment of rat cardiac allograftswith anti-Ang2 antibody (MEDI1/5) prevented the development of acuterejection in 10 days and cardiac fibrosis and allograft vasculopathy inrat cardiac allografts 8 weeks after transplantation. (A-C) Shows thedensity of VCAM-1+, ICAM-1+, and P-selectin+ microvascular vessels inmyocardial cross sections 10d after transplantation. (D-H) Shows thenumbers of intragraft ED1+ macrophages, MPO+ neutrophils, CD4+ T cells,CD8+ T cells, and OX62+ dendritic cells 10d after transplantation. n=6per group. (I-N) The relative mRNA expression of Ang2, IL-6, IFNγ,ICAM-1, CCL3, and TGFβ in allografts 10d after transplantation. (O) Thesurvival of allografts in chronic rejection experiments. Data are givenby Kaplan-Meier survival plot, or by box plots showing the upper extreme(excluding outliers), upper quartile, median, lower quartile, and lowerextreme (excluding outliers). *P<0.05, **P<0.01 using the Mann-Whitney Utest.

FIG. 10 shows cardiac allograft recipient treatment with anti-Ang2antibody (MEDI1/5) does not prevent microvascular leakage or no-reflowphenomenon, but did prevent allograft myocardial injury, endothelialactivation and inflammation 6 h after cardiac transplantation. Therecipients received anti-Ang2 antibody (MEDI1/5) 4 h prior to thetransplantation (1 mg/kg; i.p.). (A) Shows microvascular leakagemeasured by quantifying myocardial absorbance of extravasated Evans Bluedye 30 min after reperfusion (modified Miles assay). (B-D) Showsmyocardial microvascular perfusion measured by quantifying the densityof endothelium-binding FITC-labeled Lycopersicon esculentum lectin(green) 30 min after the reperfusion. (E) Shows the recipient serumlevels of cardiomyocyte specific troponin T (TnT) 6 h after reperfusion.(F-G) Shows the density of VCAM-1+ and ICAM-1+. (H-L) Shows the numbersof intragraft ED1+ macrophages, MPO+ neutrophils, CD4+ T cells, CD8+ Tcells, and OX62+ dendritic cells 6 h after reperfusion. n=6 per group.Magnification 100×, inset 400×; scale bars=50 μm (C and D). Data aregiven by box plots showing the upper extreme (excluding outliers), upperquartile, median, lower quartile, and lower extreme (excludingoutliers). *P<0.05, **P<0.01, using the Mann-Whitney U test.

FIG. 11 shows cardiac allograft recipient treatment with multiple dosesof anti-Ang2 antibody (MEDI1/5) prolongs allograft survival. (A) Showsthe effect of single preoperative i.p. dose of anti-Ang2 antibody (1mg/kg; black arrow) or IgG i.p. on allograft survival (n=6 per group).(B) Shows the effect multiple doses of anti-Ang2 antibody (1 mg/kg;black arrows) or IgG i.p. on allograft survival (n=12/group). Singledose was given 4 h before transplantation (A), and further consecutivedoses were given on days 1, 3, and 5 after the transplantation (B). Therecipients also received low-dose CyA (1 mg/kg/d s.c.). Data are givenby Kaplan-Meier survival plot. ***P<0.001 using the Log-rank survivalanalysis.

FIG. 12 shows cardiac allograft recipient treatment with multiple dosesof anti-Ang2 antibody (MEDI1/5) prevents the development of acuterejection and cardiac fibrosis and allograft vasculopathy. (A-C) Showsthe density of VCAM-1+, ICAM-1+, and P-selectin+ microvascular vesselsin myocardial cross sections 10 days after transplantation. (D-H) Showsthe numbers of intragraft ED1+ macrophages, MPO+ neutrophils, CD4+ Tcells, CD8+ T cells, and OX62+ dendritic cells 10d aftertransplantation. n=6 per group. (I) Shows the survival of allografts inchronic rejection experiments. (J-L) Shows the numbers of RECA-1+stained vessels 8 weeks after transplantation. (M-O) Shows the relativefibrotic area in Masson's trichrome stained mid-cardiac cross sections 8weeks after transplantation. (P-R) Shows the degree of luminal occlusionin Hematoxylin-eosin and Resorcin-Fuchsin stained mid-cardiac crosssections 8 weeks after transplantation. n=6/group (A-H); n=7 per group(I-R). Magnification 100×, inset 400×; scale bars=50 μm (K, L, N, O, Q,and R). Data are given by Kaplan-Meier survival plot, or by box plotsshowing the upper extreme (excluding outliers), upper quartile, median,lower quartile, and lower extreme (excluding outliers). *P<0.05,**P<0.01 using the Mann-Whitney U test.

FIG. 13 shows ex vivo intracoronary treatment of rat cardiac allograftswith anti-Ang2 antibody prevents myocardial injury and activation ofinnate immunity 6 h after transplantation. (A) Shows the dose-responseanalysis of the recipient serum levels of TnT 6 h after transplantationof anti-Ang2 antibody-perfused allografts, and of add-back experimentswith combination of anti-Ang2 antibody (30 ng) and recombinant Ang2 (2μg). (B) Shows the density of P-selectin+ microvascular vessels inmyocardial cross sections 6 h after reperfusion. (C-G) Shows the numbersof intragraft ED1+ macrophages, MPO+ neutrophils, CD4+ T cells, CD8+ Tcells, and OX62+ dendritic cells 6 h after reperfusion. (H-P) Shows therelative mRNA expression of HAS1-3, NF-kB, IL-1b, IL-2, IL-12p35,VEGF-A, and TGFb in allografts 6 h after transplantation. n=8 or9/group. Data are given by box plots showing the upper extreme(excluding outliers), upper quartile, median, lower quartile, and lowerextreme (excluding outliers). *P<0.05, **P<0.01, ***P<0.001 using theMann-Whitney U test.

FIG. 14 shows that adding recombinant Ang2 to ex vivo intracoronaryperfusion solution abrogates the protective effects of anti-Ang2antibody 6 h after reperfusion. (A-B) Shows the density of VCAM-1+ andICAM-1+ vessels in myocardial cross sections 6 h after reperfusion.(C-G) Shows the numbers of intragraft ED1+ macrophages, MPO+neutrophils, CD4+ T cells, CD8+ T cells, and OX62+ dendritic cells 6 hafter reperfusion. n=6 per group. Data are given by box plots showingthe upper extreme (excluding outliers), upper quartile, median, lowerquartile, and lower extreme (excluding outliers). *P<0.05, **P<0.01using Kruskall-Wallis test with Dunn correction.

DETAILED DESCRIPTION OF THE INVENTION

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an anti-Ang2 antibody” is understood torepresent one or more anti-Ang2 antibodies. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

Angiopoietin-2

Many studies of Angiopoietin-2 (“Ang2” or “Ang-2”) function havesuggested a complex situation. Ang-2 might be a complex regulator ofvascular remodeling that plays a role in both vessel sprouting andvessel regression. Supporting such roles for Ang-2, expression analysesreveal that Ang-2 is rapidly induced, together with VEGF, in adultsettings of angiogenic sprouting, whereas Ang-2 is induced in theabsence of VEGF in settings of vascular regression (Holash et al.,Science 284:1994-98 (1999); Holash et al., Oncogene 18:5356-62 (1999)).Consistent with a context-dependent role, Ang-2 binds to the sameendothelial-specific receptor, Tie-2, which is activated by Ang-1, buthas context-dependent effects on its activation (Maisonpierre et al.,Science 277:55-60 (1997)). In recent years Angiopoietin-1,Angiopoietin-2 and/or Tie-2 have been proposed as possible anti-cancertherapeutic targets (See, for example, U.S. Pat. Nos. 6,166,185,5,650,490, 5,814,464, US Patent Publ. No. 20060018909 and Int. Publ.Nos. WO 2006/068953 and WO 2007/068895).

Angiogenesis is the process of forming new capillaries from preexistingblood vessels and is an essential component of embryogenesis, normalphysiological growth, repair, and tumor expansion. Although a variety offactors can modulate endothelial cell (EC) responses in vitro and bloodvessel growth in vivo, only vascular endothelial growth factor (VEGF)family members and the angiopoietins are believed to act almostexclusively on vascular ECs (Yancopoulos et al., Nature 407:242-48(2000)).

The endothelium destabilizing properties of Ang-2 include induction ofvascular leakage by promoting EC apoptosis and by disrupting EC-ECjunctions (26). Ang-2 mediates endothelial dysfunction in various tissuestress conditions, such as airway inflammation, sepsis, hypoxia and inexperimental ischemic heart and brain models (20, 27, 28). However,exogenous and endogenous Ang-2 have different effects (29), and theirrole in IRI is not fully understood. The IRI in an allograft is followedby early infiltration of innate immune inflammatory cells, mainlymacrophages and neutrophils (30, 31). Cellular adhesion molecules playessential role in recruiting inflammatory cells to the site of injury.Ang1 inhibits leukocyte recruitment after LPS-induced endotoxicity andby preventing VEGF-induced endothelial adhesion molecule expression(32,33). In an acute kidney injury model, Kim et al. describe decreaseof the ICAM-1 and VCAM-1 expression after therapy with a cartilageoligomeric matrix protein-Ang1 fusion (9). Ang-2 inhibition has beenshown to decreases immunoreactivity of adhesion molecules in an ischemicmouse hind limb model (29). Furthermore, in a recent study withAng2-overexpressing endothelial cells, Scholz et al. showed that Ang-2has the capability of inducing inflammation and myeloid cell activationper se (1).

Antibodies

Embodiments described herein relate to methods of treatment usingantibodies or antigen-binding fragments thereof specific for Ang-2,e.g., antibodies which can be derived from the antibody 3.19.3 (see,e.g., Int. Publ. No. WO 2009/097325) and which exhibit enhancedstability and/or production efficiencies, e.g., “MEDI1/5.” In oneembodiment the methods of the invention are directed to use of anantibody or antigen-binding fragment thereof comprising the six CDRs ofanti-Ang2 antibody 3.19.3. The variable heavy and variable light chainamino acid sequences for anti-Ang2 antibody 3.19.3 are shown below.

3.19.3 light chain (SEQ ID NO: 1):EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRLLI CGASSWATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

The double underlined sequences represent VL CDRs 1-3, respectively. Theunderlined/bold residue in the 3.19.3 light chain sequence represents anunpaired cysteine (C49) that can be changed to any other amino acid.Examples of such changes are described in Int. Publ. No. WO 2009/097325.

3.19.3 heavy chain (SEQ ID NO: 2): QVQLVESGGGVVQPGRSLRLSCAASGFTFTNYGMHWG RQAPGKGLEWVA VISHDGNNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGIDFWSGLNWFDPWGQGTLVTVSS

The double underlined sequences represent VH CDRs 1-3, respectively. Theunderlined/bold residue in the 3.19.3 heavy chain sequence represents anexample of a residue that can be “backmutated” to another residue. Oneexample of such a “backmutation” is represented in the “MEDI5” heavychain sequence disclosed in Int. Publ. No. WO 2009/097325.

In some embodiments, the antibodies or antigen-binding fragments thereofbind to endogenous Ang-2 and inhibit the binding of Ang-2 to itsreceptor, Tie2. Other embodiments disclosed herein include fully humananti-Ang2 antibodies or antigen-binding fragments thereof, and antibodypreparations that are therapeutically useful. Such anti-Ang2 antibodypreparations have desirable therapeutic properties, including strongbinding affinity for Ang-2, the ability to neutralize Ang-2 in vitro,and the ability to inhibit ischemia in Ang-2-expressing cells in vivo.In certain embodiments, the antibodies or antigen-binding fragmentsthereof disclosed herein comprise the ability to specifically bind Ang-2and inhibit ischemia and/or reduce tissue inflammation. Mechanisms bywhich this can be achieved can include, but are not limited to,inhibition of binding of Ang-2 to its receptor Tie2, inhibition of Ang-2induced Tie2 signaling, or increased clearance of Ang-2, thereinreducing the effective concentration of Ang-2.

In certain embodiments, an anti-Ang2 antibody or antigen-bindingfragment thereof for use in the methods described herein is derived froman anti-Ang2 antibody which is described in Int. Appl. No. WO2009/097325, WO 2007/068895, or WO 2006/068953, each of which areincorporated herein by reference in their entireties. In one embodiment,the anti-Ang2 antibody or antigen-binding fragment thereof for use inthe methods described herein is derived from anti-Ang2 antibody 3.19.3,e.g., MEDI1/5.

One aspect of the disclosure is directed to stabilized anti-Ang2antibodies or antigen-binding fragments thereof for use in the methodsdescribed herein which comprise a substitution of an amino acid atposition 49 (as compared to the light chain variable amino acid sequenceof Ang-2 antibody 3.19.3, see SEQ ID NO: 1) as defined by the Kabatnumbering system (Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991),vols. 1-3). In one embodiment, the amino acid substitution at position49 can be any amino acid. In a specific embodiment, the amino acidsubstitution at position 49 is selected from the group consisting ofAsp, Thr, Asn, and Ala.

In another embodiment, the antibodies or antigen-binding fragmentsthereof for use in the methods described herein further comprises a Valsubstitution at position 37 of the heavy chain (as compared to the heavychain variable amino acid sequence of Ang-2 antibody 3.19.3, see, SEQ IDNO: 2) as defined by the EU numbering system (Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, NIH Publication91-3242, Bethesda Md. (1991), vols. 1-3).

In one embodiment, the antibodies or antigen-binding fragments thereoffor use in the methods described herein exhibit enhanced stability ascompared to antibody 3.19.3. In another embodiment, the antibodies orantigen-binding fragments thereof disclosed herein exhibit enhancedproduction yields as compared to 3.19.3.

In another embodiment, the antibodies or antigen-binding fragmentsthereof disclosed herein can comprise a variable light chain amino acidsequence selected from the group consisting of MEDI1 (SEQ ID NO: 3),MEDI2 (SEQ ID NO: 4), MEDI3 (SEQ ID NO: 5), MEDI6 (SEQ ID NO: 8), andMEDI4 (SEQ ID NO: 6). In another embodiment, the antibodies orantigen-binding fragments thereof disclosed herein can comprise theheavy chain variable amino acid sequence of MEDI5 (SEQ ID NO: 7). Inanother embodiment, the antibodies or antigen-binding fragments thereofdisclosed herein can comprise variable light chain sequences selectedfrom the group consisting of MEDI1 (SEQ ID NO: 3), MEDI2 (SEQ ID NO: 4),MEDI3 (SEQ ID NO: 5), MEDI6 (SEQ ID NO: 8), and MEDI4 (SEQ ID NO: 6) andfurther comprise the heavy chain variable sequence of MEDI5 (SEQ ID NO:7).

In another embodiment, the antibodies or antigen-binding fragmentsthereof for use in the methods described herein can comprise variablelight chain acid sequences selected from the group consisting of MEDI1(SEQ ID NO: 3), MEDI2 (SEQ ID NO: 4), MEDI3 (SEQ ID NO: 5), MEDI6 (SEQID NO: 8), and MEDI4 (SEQ ID NO: 6), but having a different amino acidsubstitution at position 49. In another embodiment, the antibodies orantigen-binding fragments thereof disclosed herein can further comprisethe heavy chain variable sequence of MEDI5 (SEQ ID NO: 7). In anotherembodiment, the antibodies or antigen-binding fragments thereofdisclosed herein can comprise variable light chain sequences selectedfrom the group consisting of MEDI1 (SEQ ID NO: 3), MEDI2 (SEQ ID NO: 4),MEDI3 (SEQ ID NO: 5), MEDI6 (SEQ ID NO: 8), and MEDI4 (SEQ ID NO: 6),but having a different amino acid substitution at position 49, andfurther comprise the heavy chain variable sequence of MEDI5 (SEQ ID NO:7).

In some embodiments, antibodies or antigen-binding fragments thereofdisclosed herein comprise a light chain which is engineered to remove atleast one O-glycosylation site. In some embodiments, antibodies orantigen-binding fragments thereof disclosed herein comprise a lightchain selected from the group consisting of MEDI1 (SEQ ID NO: 3), MEDI2(SEQ ID NO: 4), MEDI3 (SEQ ID NO: 5), and MEDI4 (SEQ ID NO: 6) whereinthe light chain further comprises an amino acid substitution at Kabatposition 59, wherein the amino acid is not proline. In a specificembodiment, antibodies or antigen-binding fragments thereof disclosedherein comprise a light chain having the sequence corresponding to MEDI6(SEQ ID NO: 8).

The variable light chain amino acid sequences MEDI1, MEDI2, MEDI3,MEDI4, and MEDI6; and the variable heavy chain amino acid sequence forMEDI5 are shown below. The double underlined sequences represent the VLand VH CDRs. The underlined residue is an example of a residue that canbe “backmutated” to another residue.

MEDI1 light chain  (SEQ ID NO: 3)EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRLLI TGASSWATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECMEDI2 light chain  (SEQ ID NO: 4)EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRLLI NGASSWATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECMEDI3 light chain  (SEQ ID NO: 5)EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRLLI DGASSWATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGECMEDI4 light chain  (SEQ ID NO: 6)EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRLLI AGASSWATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECMEDI6 light chain  (SEQ ID NO: 8)EIVLTQSPGTLSLSPGERATLSCRASQSITGSYLAWYQQKPGQAPRLLI T GASSWATGI ADRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECMEDI5 heavy chain  (SEQ ID NO: 7) QVQLVESGGGVVQPGRSLRLSCAASGFTFTNYGMHW VRQAPGKGLEWVA VISHDGNNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGIDFWSGLNWFDPWGQGTLVTVSS

In certain embodiments, the methods disclosed herein are for use with anantibody comprising the three variable light chain (VL) CDRs of MEDI1(SEQ ID NO: 3), MEDI2 (SEQ ID NO: 4), MEDI3 (SEQ ID NO: 5), MEDI4 (SEQID NO: 6), or MEDI6 (SEQ ID NO: 8) and the three variable heavy chain(VH) CDRs of MEDI5 (SEQ ID NO: 7). In certain embodiments, the methodsdisclosed herein are for use with an antibody comprising the VL sequenceof MEDI1 (SEQ ID NO: 3), MEDI2 (SEQ ID NO: 4), MEDI3 (SEQ ID NO: 5),MEDI4 (SEQ ID NO: 6), or MEDI6 (SEQ ID NO: 8) and the VH sequence ofMEDI5 (SEQ ID NO: 7). In some embodiments, the antibody of the methodsdisclosed herein is MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, or MEDI4/5.

In one embodiment the anti-Ang2 antibody or antigen-binding fragmentthereof is an antagonist of the biological activity of Angiopoietin-2.In further embodiments, the antagonist of Angiopoietin-2 is a monoclonalantibody. In yet further embodiments, the antagonist of Angiopoietin-2is a fully human monoclonal antibody. In some embodiments the fullyhuman monoclonal antibody is selected from any one of: 3.31.2, 5.16.3,5.86.1, 5.88.3, 3.3.2, 5.103.1, 5.101.1, 3.19.3, 5.28.1, 5.78.3,MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, or MEDI4/5, which are disclosed inInt. Publ. No. WO 2009/097325. In some embodiments the anti-Ang2antibody disclosed herein comprises the six CDRs of an antibody selectedfrom any one of: 3.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2, 5.103.1,5.101.1, 3.19.3, 5.28.1, 5.78.3, MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, orMEDI4/5. In further embodiments, the fully human monoclonal antibodybinds to the same epitope as any one of the following fully humanmonoclonal antibodies: 3.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2, 5.103.1,5.101.1, 3.19.3, 5.28.1, or 5.78.3, which are disclosed in Int. Appl.No. WO 2006/068953. In another aspect, the antibody of any of themethods disclosed herein binds to the same epitope as any one or more ofthe following antibodies: the MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, orMEDI4/5.

The invention also provides antibodies that competitively inhibitbinding of an antibody to a Ang-2 epitope as determined by any methodknown in the art for determining competitive binding, for example, theimmunoassays and antibody binding assays described herein. In certainaspects, the antibody competitively inhibits binding to the epitope byat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50%. In certain aspects, theantibody of the invention competitively inhibits binding of MEDI1/5,MEDI2/5, MEDI3/5, MEDI6/5, or MEDI4/5 to Ang-2.

Methods of Treatment Using Anti-Ang2 Antibodies

Terms such as “treating” or “treatment” or “to treat” or “ameliorating”or “alleviating” or “to alleviate” can refer to both 1) therapeuticmeasures that cure, slow down, lessen symptoms of, reverse, and/or haltprogression of a diagnosed pathologic disease, condition or disorder ina subject or tissue in vivo or ex vivo; and 2) prophylactic orpreventative measures that prevent, reduce the risk of, and/or slow thedevelopment of a targeted pathologic disease, condition or disorder in asubject or tissue in vivo or ex vivo. Thus, a subject or tissue in needof treatment can include a subject or tissue already diagnosed with thedisease, condition or disorder; a subject or tissue prone to have thedisease, condition or disorder; and a subject or tissue for which thedisease, condition or disorder is to be prevented. Beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of the disease, condition or disorder;stabilized (i.e., not worsening) state of the disease, condition ordisorder; delay or slowing of the progression of the disease, conditionor disorder; amelioration or palliation of the disease, condition ordisorder, and remission (whether partial or total), whether detectableor undetectable. “Treatment” can also mean prolonging survival ascompared to expected survival if not receiving treatment. A subject ortissue in need of treatment include a subject or tissue alreadydiagnosed with the disease, condition or disorder as well as a subjector tissue prone to have the disease, condition or disorder; or a subjector tissue in which the disease, condition or disorder is to beprevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. In one aspect, the subject is a human.

By “tissue,” is meant any body tissue or organ, e.g., heart, kidney,brain, smooth muscle or intestine tissue, particularly from a mammaliansubject, for which diagnosis, prognosis, or therapy is desired. Incertain embodiments, the tissue is an ischemic tissue, i.e., the tissuehas undergone a period of ischemia or lack of oxygen. In certainembodiments, the ischemic tissue disclosed herein is injured by surgery,for example, by coronary artery bypass grafting, correction of acongenital heart defect, replacement of a heart valve, or hearttransplantation.

In one embodiment, the tissue is an allograft. As used herein“allograft” is a tissue (e.g., solid organ) graft from a donor of thesame species as the recipient but not genetically identical. In anotherembodiment, the tissue is an autograft. As used here an “autograft” is atissue (e.g., bone, skin or blood vessels) that is transplanted withinthe same person's body.

Aspects disclosed herein include methods of using anti-Ang2 antibodiesor antigen-binding fragments thereof for treating diseases orconditions, e.g., IRI, in a subject or tissue. In certain embodiments,the methods disclosed herein include using anti-Ang2 antibodies orantigen-binding fragments for modifying Ang-2 levels or Ang-2-mediatedactivities in a subject or tissue. For example, anti-Ang2 antibodies orantigen-binding fragments thereof are useful for preventing or reducingAng-2 mediated Tie2 signal transduction. The mechanism of action of thisinhibition can include inhibition of Ang-2 from binding to the receptorTie2; inhibition of Ang-2 induced Tie2 signaling; Ang-2 mediatedphosphorylation of Tie-2; or enhanced clearance of Ang-2, therebylowering the effective concentration of Ang-2 for binding to Tie-2. Inone embodiment, the anti-Ang2 antibody or antigen-binding fragmentthereof reduces expression of VEGF, TGFβ, or both (e.g., for treatmentof diseases, disorders or conditions associated with inflammation,vascular leakage or fibrosis). In another embodiment, the anti-Ang2antibodies or antigen-binding fragments thereof can act through reducingcirculating Ang-2 levels. In one embodiment, anti-Ang2 antibodies orantigen-binding fragments thereof can inhibit Ang-2 function in anischemic molecular signaling cascade. In yet another embodiment,anti-Ang2 antibodies or antigen-binding fragments thereof are useful forinhibiting an IRI-induced inflammatory response.

Diseases, disorders or conditions that are treatable with an anti-Ang2antibody or antigen-binding fragment thereof disclosed herein (e.g.,MEDI1/5), e.g., through one or more of the inhibition/reductionmechanisms disclosed herein, include, but are not limited to, ischemicreperfusion injury (IRI) of any organ or tissue or multiple organs, suchas heart (e.g., acute coronary syndrome), kidney (e.g., acute kidneyinjury), intestine (e.g., intestitial ischemia and multiorgan failure),brain (e.g., stroke, see, e.g., Liu et al., JBC 284(34):22680-22689(2009)); any type of vascular stroke; trauma and resuscitation (e.g.,multiorgan failure, acute kidney injury, intestinal injury); orcirculatory arrest (e.g., hypoxic brain injury, multiorgan failure,acute kidney injury). In another embodiment, the disease, disorder orcondition is ischemia and/or reperfusion associated with major surgeryin which the target organ or tissue is ischemic before, during, or aftersurgery, such as cardiac surgery (e.g., acute heart failure aftercardiopulmonary bypass); thoracic surgery (e.g., acute lung injury);peripheral vascular surgery (e.g., compartment syndrome of extremity);major vascular surgery (e.g., acute kidney injury); and solid organtransplantation (e.g., acute graft failure, early graft rejection). Inanother embodiment, the disease, disorder or condition is a fibrotic orhypoxic condition (e.g., pulmonary fibrosis or lung hypoxia). In anotherembodiment, the disease, disorder or condition is accelerated orallograft artheriosclerosis. In another embodiment, the disease,disorder or condition is resuscitation of a subject, e.g., wherecirculatory collapse is followed by vascular leakage, e.g., in thelungs. In one embodiment, the subject has acute respiratory distresssyndrome (ARDS) (see, e.g., Parikh, et al., PLOS Medicine 3(3):356-370(2006)). In another embodiment, the disease, disorder or condition issepsis, posttraumatic shock or hypoxic shock. Further embodimentsdisclosed herein include treatment of solid organ allografts in warmblood perfusion systems. Further embodiments disclosed herein alsoinclude treatment or prevention of chronic rejection of transplantedtissue, e.g., cardiac allografts. In certain embodiments, the subject tobe treated by the methods disclosed herein has increased circulatingAng-2. In certain embodiments, the methods of treatment disclosed hereincan be preceded by a diagnostic test. In certain embodiments, thedisease, disorder or condition disclosed herein is diagnosed bymeasuring Ang2 levels in the subject, e.g., by detecting circulatingAng2 with an anti-Ang2 antibody disclosed herein.

In the broadest sense, ischemic reperfusion injury or ischemia andreperfusion-elicited tissue injury (“IRI”) is the tissue damage causedwhen blood supply returns to the tissue after a period of ischemia orlack of oxygen. The absence of oxygen and nutrients from blood duringthe ischemic period creates a condition in which the restoration ofcirculation results in inflammation and oxidative damage through theinduction of oxidative stress. IRI contributes to morbidity andmortality in a wide range of pathologies, e.g, single organ ischemia andreperfusion (e.g., of the heart, kidney, intestine and brain);multiple-organ ischemia and reperfusion (e.g., due to trauma andresuscitation, circulatory arrest, sickle cell disease, and sleepapnea); ischemia and reperfusion during major surgery (e.g., cardiacsurgery, thoracic surgery, peripheral vascular surgery and solid organtransplantation). See, e.g. Eltzschig and Eckle, Nature Medicine17(11):1391-1401 (2011). In certain embodiments, the subject suffersfrom one or more of the pathologies disclosed herein.

In addition or further to the pre-clinical models disclosed in detailherein, the additional diseases or disorders can be tested using modelsaccepted in the art, e.g., for myocardial ischemia/reperfusion (e.g.,Shyu et al., Clinical Science 105:287-294 (2003)); accelerated orallograft artheriosclerosis (e.g., Libby et al., Circulation107:1237-1239 (2003)) and Nykanen et al., Circulation 107:1308-1314(2003)); pulmonary leakage and congestion or ischemic pulmonary fibrosis(e.g., Parikh, et al., PLOS Medicine 3(3):356-370 (2006)); skin DelayedType Hypersensitivity (DTH) (e.g., Dhabhar F S, Brain Behav Immun.16(6):785-98 (2002)); kidney ischemia and kidney transplantation (e.g.,Jung Y, et al., Am J Physiol Renal Physiol. 297(4):F952-60 (2009) andSalahudeen A K, Am J. Physiol Renal Physiol 287:F181-F187 (2004)); andischemic stroke (e.g., Engel et al., J. Vis. Exp. (47), e2423, DOI:10.3791/2423 (2011)).

In certain embodiments, the IRI disclosed herein is due to tissuetransplantation (e.g., transplantation of a solid organ such as heart,kidneys, liver, lungs, pancreas, or intestine). In other embodiments,the IRI is due to cardiothoracic, vascular or general surgery;myocardial infarction; ischemic stroke; acute kidney injury; trauma orresuscitation; circulatory arrest; collapsed lung; sickle cell diseaseor sleep apnea.

One aspect disclosed herein is directed to a method for treating anIRI-induced inflammatory response in a subject or tissue comprisingadministering a therapeutically effective amount of an anti-Ang2antibody or antigen-binding fragment thereof for use in the methodsdescribed herein to the subject or tissue in need thereof.

IRI is associated with disruption of vascular endothelial integrity, ECdamage and subsequent pathological remodeling leading to chronicrejection. In one embodiment, the anti-Ang2 antibody or antigen-bindingfragment thereof is administered to a tissue, e.g., intracoronaryadministration to a heart, to treat or protect the tissue from IRI.

Chronic rejection of cardiac allograts manifests as fibroblastproliferation, collagen deposition and vascular occlusion throughout theallograft, generally described as cardiac allograft vasculopathy (CAV).CAV manifests as allograft dysfunction, diastolic insufficiency andleads inevitably to graft failure. Graft failure is the main limitingfactor of cardiac transplantation patient's survival (35). After decadesof research, the pathophysiology of chronic rejection still remainssomewhat unknown. Scientists and clinicians agree that the role ofTh1-type T-cell mediated sustained inflammation as the main drivingforce towards chronic rejection. Thus, current clinicalimmunosuppressive strategies inhibit acute and chronic rejectiondevelopment with steroids and IL-2 signaling targeted calcineurininhibitors, such as cyclosporin A and tacrolimus, antimetabolites, andcorticosteroids (36). However, even with sufficient immunosuppressionpreventing episodes of acute rejection, the development of CAV is stilla significant survival limiting factor of cardiac allograft recipients(1). Emerging evidence suggest crucial role of IRI and innate immuneresponses in the activation of adaptive immune responses (37). IRI ofthe allograft exposes endogenous DAMPs, that are recognized by TLRs onantigen presenting cells, such as macrophages and DCs, resulting in DCmaturation and migration to secondary lymphatic organs, where antigensare presented to naïve Th1-type T-cells (22). In addition, earlymicrovascular dysfunction of cardiac, lung and renal allografts inflictsprimary graft dysfunction and predicts the development of subsequentpathological changes (2. Hollenberg S M et al. Circulation. 2001;104(25):3091-3096; Luckraz H et al. J Heart Lung Transplant. 2004;23(5):527-531; Ishii Y et al. Transplant Proc. 2005; 37(2):981-983).

Another aspect is directed to a method for treating an IRI-inducedmicrovascular dysfunction, cardiac fibrosis and/or chronic allograftvasculopathy (CAV) in a subject or tissue comprising administering atherapeutically effective amount of an anti-Ang2 antibody orantigen-binding fragment thereof described herein to the subject ortissue in need thereof. In certain embodiments, the anti-Ang2 antibodyor antigen-binding fragment thereof is used for primary allograftprotection. In certain embodiments, the anti-Ang2 antibody orantigen-binding fragment thereof is used in a perfusion solution formaintaining the endothelial integrity of the coronaries during thetransplantation surgery.

In certain methods, the anti-Ang2 antibody or antigen-binding fragmentthereof for use in the methods described herein are used to treatdiseases associated with an inflammatory response.

Also disclosed herein are methods for reducing microvascularpermeability in a tissue, increasing microvascular perfusion in atissue, and/or reducing inflammation in a tissue comprisingadministering a therapeutically effective amount of an anti-Ang2antibody or antigen-binding fragment thereof to a tissue in needthereof.

In another aspect disclosed herein, the method is directed to treatingor ameliorating myocardial ischemia, comprising administering atherapeutically effective amount of an anti-Ang2 antibody orantigen-binding fragment thereof to a subject in need thereof. Anotheraspect of the disclosure is a method of protecting a solid organtransplant tissue, comprising administering an effective amount of ananti-Ang2 antibody or antigen-binding fragment thereof to an allograft.In yet another aspect, the method is directed to treating or preventingchronic rejection, comprising administering a therapeutically effectiveamount of an anti-Ang2 antibody or antigen-binding fragment thereof to apatient in need thereof.

The compositions and methods disclosed herein can be used with one ormore conventional therapies that are used to prevent, manage or treatany of the above diseases or disorders.

In one embodiment, the anti-Ang2 antibody or antigen-binding fragmentthereof for use in the methods described herein is administered as amonotherapy. In another embodiment, the anti-Ang2 antibody orantigen-binding fragment thereof for use in the methods described hereinis administered in combination with other therapies. In one embodiment,the anti-Ang2 antibody or antigen-binding fragment thereof for use inthe methods described herein is coadministered with one or moreadditional (or second) agents at the same time or a different time. Inone embodiment, the method or use disclosed herein can be coadministeredwith an anti-inflammatory agent and/or an immunosuppressive agent, e.g.,steroids; a biologic agent (see, e.g., Bann and Gaston, American Journalof Transplantation 11:681-686 (2011)), e.g., Belatacept, a CD20 or CD19targeting antibody (e.g., Rituximab), a TNF targeting agent; or acalcineurin inhibitor, such as cyclosporin, tacrolimus, mycophenolicacid, azathioprine, statins, anti-thymocyte globuline, everolimus, orsirolimus.

Such conjoint or coadministered treatments can be achieved by way of thesimultaneous, sequential or separate dosing of the individual componentsof the treatment. Such combination therapies can employ the antibodiesor antigen-binding fragments thereof for use in the methods disclosedherein within the dosage range described herein and the otherpharmaceutically active agent within its approved dosage range.

In another aspect, the methods are directed to administration of acomposition, for example, but not limited to, a pharmaceuticalcomposition, containing one or more anti-Ang2 antibodies orantigen-binding fragments thereof for use in the methods describedherein, formulated together with a pharmaceutically acceptable carrier.

The methods disclosed herein can be formulated according to well-knownmethods (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of an anti-Ang2 antibody or antigen-binding fragmentthereof for use in the methods described herein that when administeredbrings about a positive therapeutic response with respect to treatmentof a subject or a tissue with the disease, condition or disorder to betreated.

Determination of the appropriate dose can be made by the clinician,e.g., using parameters or factors known or suspected in the art toaffect treatment or predicted to affect treatment. Generally, the dosebegins with an amount somewhat less than the optimum dose and it isincreased by small increments thereafter until the desired or optimumeffect is achieved relative to any negative side effects. Importantdiagnostic measures include those of symptoms of, e.g., the inflammationor level of inflammatory cytokines produced.

Compositions comprising anti-Ang2 antibody or antigen-binding fragmentthereof for use in the methods described herein can be provided as asingle dose to a tissue (e.g., by perfusion) or subject (e.g., bycontinuous infusion or doses at intervals of, e.g., once daily, onceweekly, or 1-7 times per week). Doses can be provided intracoronarily toa tissue, e.g., by perfusion to an allograft. A specific dose protocolis one involving the maximal dose or dose frequency that avoidssignificant undesirable side effects and can be determined by a personof ordinary skill in the art.

A total single, daily or weekly dose can be at least 0.05 μg/kg to atleast 50 mg/kg (see, e.g., Yang, et al. (2003) New Engl. J. Med.349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698; Liu,et al. (1999) J. Neural. Neurosurg. Psych. 67:451-456; Portielji, et al.(2003) Cancer Immunol. Immunother. 52:133-144). The single, daily orweekly dose can be at least 15 μg to 100 μg. The doses administered to asubject or tissue can number at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12, or more.

The dosage of the anti-Ang2 antibody or antigen-binding fragment thereoffor use in the methods described herein that is administered to asubject can be 0.0001 mg/kg to 100 mg/kg of the patient's body weight.The dosage of the anti-Ang2 antibody or antigen-binding fragment thereoffor use in the methods described herein can be calculated using thesubject's weight in kilograms (kg) multiplied by the dose to beadministered in mg/kg. The dosage of anti-Ang2 antibody orantigen-binding fragment thereof for use in the methods described hereincan be 150 ng/kg or less, 50 μg/kg or less, 25 μg/kg or less, 10 μg/kgor less, 5 μg/kg or less, 1 μg/kg or less, 0.5 μg/kg or less, or 0.5μg/kg or less of a patient's body weight.

Unit dose of the anti-Ang2 antibody or antigen-binding fragment thereoffor use in the methods described herein can be 0.1 mg to 20 mg.

The dosage of the anti-Ang2 antibody or antigen-binding fragment thereoffor use in the methods described herein can achieve a serum titer of atleast 0.1 μg/ml in a subject. Alternatively, the dosage of the anti-Ang2antibody or antigen-binding fragment thereof for use in the methodsdescribed herein can achieve a serum titer of at least 0.1 μg/ml in thesubject.

An effective amount for a particular subject or tissue can varydepending on factors such as the condition being treated, the overallhealth of the subject, the method route and dose of administration andthe severity of side effects (see, e.g., Maynard, et al. (1996) AHandbook of SOPs for Good Clinical Practice, Interpharm Press, BocaRaton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice,Urch Publ., London, UK).

In certain embodiments, an allograft is perfused with an anti-Ang2antibody or antigen-binding fragment thereof for use in the methodsdescribed herein. In one embodiment, the anti-Ang2 antibody orantigen-binding fragment thereof for use in the methods described hereinis administered preoperatively. In some embodiments, the administrationof an anti-Ang2 antibody or antigen-binding fragment thereof is prior tothe removal of a graft from the donor (e.g., to the donor or donortissue), prior to the transplantation of the graft to the recipient(e.g., to the donor tissue after removal from the donor, e.g., byperfusion of the organ), at the time of tissue reperfusion, aftertransplantation to the recipient (e.g., after reperfusion), or anycombination thereof.

Another embodiment disclosed herein includes an assay kit for treatingany of the diseases or disorders disclosed herein comprising ananti-Ang2 antibody or antigen-binding fragment thereof for use in themethods described herein.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety.

EXAMPLES I. Materials and Methods

Cell Cultures.

To analyze the effects of anti-Ang2 antibodies in hypoxic endothelialcells, human dermal blood microvascular endothelial cells (BECs)(PromoCell, Heidelberg, Germany) were maintained on fibronectin-coateddishes in Endothelial Cell Basal Medium MV (ECBM, PromoCell) with growthsupplements provided by the manufacturer and used in passages 2-6.Hypoxic treatment (1% O2) was carried out in an Invivo2 400 hypoxiachamber (Ruskinn Technology, Bridgend, UK). Tie2-GFP retroviraltransfected cells were produced as described in (Augustin et al., NatRev Mol Cell Biol. 2009; 10:165-177). Cells were treated withnon-specific IgG (2 μg/ml) or anti-Ang2 antibodies (MEDI1/5) (2 μg/ml).The cells were fixed, permeabilized and stained for Ang2 and Tie2.

Transmission Electron Microscopy (TEM).

The effects of preservation prior to the transplantation were analyzedby recovering naïve Dark Agouti (DA, RT1av1) rat hearts and perfusingthe coronaries with 200 μl of anti-Ang2 antibody (MEDI1/5; 150 ng/ml, inPBS) or non-specific IgG (150 ng/ml, in PBS). Next, the hearts weresubjected to 4-h cold preservation, following 1-h warm preservation inroom temperature. The samples of the mid-cardiac specimens werecollected and fixed with 2.5% glutaraldehyde in phosphate buffer (pH7.4). The Advanced Microscopy Unit (Haartman Institute, University ofHelsinki, Helsinki, Finland) prepared the samples by TEM-routine:samples were post-fixed in 1% osmium tetroxide, then dehydrated ingraded ethanol, and embedded in Epoxy resin LX-112. Ultra-thin sectionswere cut at around 80 nm. Electron staining was performed with uranylacetate and lead citrate in Leica EM STAIN automatic stainer. Thesections were examined with JEM-1400 TEM (JEOL, Tokyo, Japan) at 80 KVand the incidence of endothelial cell (EC)-EC junction gaps and ECblebbing were analyzed with 25000× magnification.

Animals and Cardiac Allograft Models.

Heterotopic cardiac transplantations between fully MHC-mismatchedpathogen-free inbred 8- to 12-week-old male Dark Agouti (DA, RT1av1)donor and Wistar Furth (WF, RT1u) recipient rats (Harlan Laboratories,Boxmeer, The Netherlands) were performed. Donor hearts were perfusedwith ice-cold heparinized phosphate-buffered saline (PBS) and thenexcised. For the ex vivo intracoronary treatment, the coronaries werethen perfused with 200 μl of anti-Ang2 antibody (MEDI1/5; 150 ng/ml, inPBS), non-specific IgG (150 μg/ml, in PBS), or recombinant Ang2 (23) (50μg/ml, in PBS). Unless stated otherwise, the allografts were subjectedto 2 to 4-h hypothermic preservation before transplantation (i.e., theallografts were preserved in +4° C. PBS (cold ischemia) instead ofimmediate transplantation). Warm ischemia was standardized to 1 h. Inthe recipient treatment groups, the recipients received anti-Ang2antibody (1 mg/kg, in PBS), or non-specific IgG (1 mg/kg, in PBS) i.p. 4h before heart transplantation. Another group also received consecutivedoses of anti-Ang2 antibody 1, 3, and 5 d after the transplantation.

The effect of cold preservation on the integrity of endothelial cellswas analyzed by transmission electron microscopy in the non-transplanteddonor hearts after 4-h cold and 1-h warm ischemia. After preservation,heterotopic cardiac transplantations were performed between fullyMHC-mismatched, pathogen-free, inbred 8- to 12-week-old male DA donorand Wistar Furth (WF, RT1u) recipient rats (Harlan Laboratories,Boxmeer, The Netherlands) (Ono K and Lindsey E S., J Thorac CardiovascSurg. 1969; 57:225-229). After reperfusion, the recipients weresacrificed at 30 min to analyze the effect on microvascular perfusionand permeability with lectin perfusion and modified Miles assay, at 6 hto analyze endothelial activation, inflammatory cell influx, myocardialinjury, and innate immune activation, and at 8 weeks to analyze theactivation of adaptive immune response and microvascular density and thedegree of cardiac fibrosis and allograft vasculopathy. To preventirreversible episodes of acute allograft rejection, to achieve long-termallograft survival, and to enable the development of chronic rejection,the recipients received s.c. cyclosporine A (Novartis, Basel,Switzerland) 2 mg/kg/day for the first 7 days and 1 mg/kg/daythereafter. All allografts were beating upon recovery. Each groupincluded six to eight animals.

The State Provincial Office of Southern Finland approved all animalexperiments. The animals received care in compliance with the Guide forthe Care and Use of Laboratory Animals as outlined by the NationalAcademy of Sciences (ISBN 0-309-05377, revised 1996). The rats inhaledisoflurane (Isofluran, Baxter, Deerfield, Ill.) in anesthesia andreceived buprenorphine (Temgesic, Schering-Plough, Kenilworth, N.J.) forperioperative analgesia.

Microvascular Leakage and Reflow.

A modified Miles Assay was used to measure extravasation of plasmaproteins from the microvasculature into the interstitial space ofcardiac allografts. Immediately after reperfusion, the recipients wereinjected (i.v.) with Evans Blue dye (Sigma-Aldrich; 30 mg/ml diluted in0.9% NaCl; n=6 or 7), which circulated for 30 min. To detect perfusedvessels 30 min after reperfusion, 50 μl FITC-labeled Lycopersiconesculentum (tomato) lectin (Vector Laboratories, Burlingame, Calif.)diluted in 150 μl of 0.9% NaCl was injected into the aortic root a fewminutes before removing the allografts. Immediately thereafter, a smallincision to the right ventricle was made and the coronary network wasretrogradely flushed with 5 ml of 1% PFA in 0.05 M citrate buffer, pH3.5. For quantification of extravasated Evans Blue, 100 mg of apicalmyocardium was dissolved into 500 μl of formamide and left on a shakerat +60° C. for 24 h. The absorbance of formamide containing dissolvedEvans Blue dye was measured by spectrophotometry at 610 nm wavelength.FITC positive microvascular vessels were quantified from mid-axial crosssections from 10 random fields of each cardiac cross section byfluorescence microscopy, and the result was given as the mean density ofperfused vessels per mm².

Acute Myocardial Injury.

Myocardial troponin T (TnT) levels in serum samples, derived at 6 hafter reperfusion, were used to determined acute myocardial injury. TnTwas analyzed with the fifth generation TnT test (Troponin T STAT, RocheDiagnostics, Mannheim, Germany), which showed cross-reactivity of 0.001%with TnT originating in skeletal muscle at a concentration of 2.000μg/ml. The functional sensitivity was 0.05 μg/1 and the lower detectionlimit 0.01 μg/l. The TnT was measured by electrochemiluminescenceimmunoassay (ECLIA) with the Elecsys 2010 immunoassay analyzer (RocheDiagnostics).

RNA Isolation and Quantitative RT-PCR.

Total RNA was isolated from tissue samples dissected from the myocardialmidpiece of the allografts according to the manufacturer's instructionsusing RNeasy kit (Qiagen, Hilden, Germany) and reverse transcribed withthe High-RNA-to-cDNA kit (Applied Biosystems, Foster City, Calif.).Quantitative real-time PCR (RT-PCR) was performed on a RotorGene-6000(Corbett Research, Doncaster, Australia) using 2× DyNAmo Flash SYBRGreen Master mix (Finnzymes, Espoo, Finland). The mRNA quantities of thefollowing factors were measured from each group: innate immune receptorsTLR2 and TLR4, their ligands biglycan, HAS1-3, HMGB1, transcriptionfactor NF-κB, dendritic cell (DC) maturation markers CD80, CD86, andCD83, inflammatory cytokines TNF-α, IL-1β, IP-10, TGF-β, IL-6 and IL-10.The number of mRNA copies of the gene of interest was calculated from acorresponding standard curve using RotorGene software. Of testedhousekeeping genes (18SRNA, GAPDH, β-actin and TBP), 18SRNA was moststably expressed and therefore all RT-PCR data were normalized against18SRNA. Primers are shown in Table 1.

TABLE 1 The genes and corresponding GenBank access numbers for theprimers used in the RT-PCR analysis. Gene Name GenBank No. HAS1NM_172323 HAS2 NM_013153 HAS3 NM_172319 Biglycan NM_017087 HMGB1NM_012963 TLR2 NM_198769 TLR4 NM_019178 NFκB L26267 IL1b NM_031512 IL2NM_053836 IL4 NM_201270 IL6 NM_012589 IL10 NM_012854 IL12p35 NM_053390TNFα NM_012675 IFNγ NM_138880 IP10 RNU22520 VEGF-A NM_031836 CD80NM_012926 CD83 NM_001108410 CD86 NM_020081 TGFβ NM_021578 KLF2NM_001007684 Ang1 NM_053546 Ang2 NM_134454 LFA-1 NM_001037780 VCAM-1NM_012889 ICAM-1 NM_012967 18SrRNA X01117 β-actin NM_031144 TBPNM_001004198 GAPDH BC087743

Immunohistochemistry and Immunofluorecence Stainings.

Cryostat sections were stained for subsets of inflammatory cells andvessels using the peroxidase ABC method (Vectastain Elite ABC Kit,Vector Laboratories) and the reaction was developed with3-amino-9-ethylcarbazole (AEC, Vectastain). Immunofluorescent stainingwas performed using Alexa 568 red and Alexa 488 green (Promega, Madison,Wis.) secondary antibodies. We used the following antibodies anddilutions: RECA-1 for endothelium (50 μg/ml, MCA97, AbD Serotec,Dusseldorf, Germany); CD4 for T cells (5 μg/ml, 22021D, BD Pharmingen,San Diego, Calif.), CD8 for T cells (5 μg/ml, 22071D BD Pharmingen), ED1for macrophages (5 μg/ml, 22451D, BD Pharmingen); MPO for neutrophils(20 μg/ml, ab9535, Abcam, Cambridge, UK); OX62 for dendritic cells (10μg/ml, MCA 1029G, Serotec, Oxford, UK); VCAM-1 (10 μg/ml, MMS-141P,Covance, Princeton, N.J.); p-adducin (phospho T445, 10 μg/ml, ab58485,Abcam). The immunoreactivity was quantified in a blinded manner using200× or 400× magnification.

Enzyme-Linked Immunosorbent Assay (ELISA).

To quantify Ang2 from human and rat serum samples, ELISA was performedaccording to the Manufacturer's instructions: human Ang2 (DANG20; R&DSystems); rat Ang2 (E90009Ra; Uscn Life Science Inc., Wuhan, China).

Histology.

Paraformaldehyde-fixed paraffin mid-cardiac cross sections were used forhistological staining. The degree of cardiac fibrosis was determinedfrom cross sections stained with Masson's trichrome staining and scoredusing computer-assisted image processing (Zeiss Axiovision 4.4, Munich,Germany) by measuring the average proportional area stained for fibrosisfrom photographs captured with 100× magnification. Chronic allograftvasculopathy was determined from cross sections stained withhematoxylin-eosin and Resorcin-Fuchsin (for internal elastic lamina) bymeasuring the area between the internal elastic lamina and vessel lumen.Arterial occlusion percentage was determined as the ratio of neointimalarea to internal elastic lamina area. Small (diameter <40 μm), medium(40-120 μm) and large arteries (>120 μm) were distinguished and analyzedseparately.

Statistics.

All data are mean±SEM by box plots showing the upper extreme (excludingoutliers), upper quartile, median, lower quartile, and lower extreme(excluding outliers) for continuous variables (SPSS Statistics 15.0;SPSS Inc., Somers, N.Y.). The outliers are shown as circles outside thebox and analyzed by Mann-Whitney U test using PASW Statistics 19.0 (SPSSInc., Chicago, Ill.). P<0.05 was considered to be statisticallysignificant.

II. Results Example 1 Hypoxia Increases Ang2 Deposition in EndothelialCell-Cell Junctions and Ex Vivo Intracoronary Perfusion with Anti-Ang2Antibody Promoted Endothelial Stability During Cold and Warm Ischemia

In cultures of human umbilical vein endothelial cells, Ang2 expressionand secretion is increased in response to hypoxia (15). In order tomodel Ang2 function in the allograft microvasculature, endothelial cellmonolayers were subjected to hypoxia for various periods of time, andAng2 was analyzed by immunofluorescent staining. The resultsdemonstrated that a 16-24 h period of hypoxic culture induced depositionof Ang2 in EC-EC junctions where Ang2 co-localized with Tie2. Anti-Ang2antibody (MEDI1/5), but not control-IgG, inhibited the deposition ofAng2-Tie2 complexes (FIG. 1). Ang2 competes with Ang1 for binding toTie2 in endothelial cell junctions, but is a weaker agonist of Tie2phosphorylation than Ang1 (Saharinen P, et al., Nat. Cell Biol. 2008;10:527-537). Thus, these results support that endogenous Ang2 hasinhibitory effects on Ang1 signaling, and that blocking endogenous Ang2may be useful in treatment of conditions originating fromischemia/hypoxia.

The effect of Ang2 neutralization on endothelial integrity was alsotested. Hypoxia induces EC blebbing—a phenomenon described by formationof cell membrane protrusions and disintegration—and promotes endothelialinstability. Hypothermic (cold) and warm preservation induces EC-EC gapformation in a cardiac allograft model (38). Transmission electronmicroscopy (TEM) imagining of non-transplanted DA hearts subjected to4-h hypothermic and 1-h warm preservation demonstrated thatintracoronary perfusion with anti-Ang2 antibody (MEDI1/5) inhibited ECblebbing and EC-EC gap formation compared to abundant blebbing and EC-ECgap formation in the control-IgG-treated allografts (FIG. 2A-D; P<0.05).The results show that inhibition of Ang2 with neutralizing antibodiesincreases endothelial stability. Furthermore, these results support thatAng2 acted as an endothelium destabilizing Ang1 antagonist, in ratcardiac allografts, and supports that Ang2 destabilizes microvascularendothelium in the heart during preservation.

Example 2 Ex Vivo Intracoronary Perfusion with Anti-Ang2 PreventedMicrovascular Permeability and No-Reflow Phenomenon During Reperfusionin Cardiac Allografts

The inhibition of endogenous Ang2 signaling with Ang2 blocking antibody(MEDI1/5) was evaluated. Microvascular leakage was analyzed with amodified Miles assay and microvascular no-reflow phenomenon was analyzedwith fluorescent lectin perfusion 30 min after reperfusion. Allograftswere intracoronarily perfused with recombinant anti-Ang2 antibody(MEDI1/5) (n=8) or IgG control (n=7); subjected to a 4-h hypothermicpreservation; and transplanted and recovered at 30 minutes afterreperfusion (after transplantation, the clamps preventing bloodflow wereopened and circulation to the allograft was re-established). As usedherein, this perfusion of ischemic organ or tissue is calledreperfusion. The allograft started beating within about a minute.Vascular permeability results were measured by quantifying Evans Blueabsorbance from the myocardium at 30 minutes after reperfusion.Capillary perfusion results were measured by quantifying tomato lectinpositive capillaries from cardiac cross sections from at least 10 randomimmunofluorescent images. The mean density of the capillaries wasmeasured by quantifying lectin positive vessels in histologicalsections. Anti-Ang2 antibody treatment was compared to controlallografts at 30 minutes after reperfusion. The Mann-Whitney U test wasused to compare anti-Ang2 and IgG treatment.

Preoperative intracoronary administration of anti-Ang2 antibody(MEDI1/5) significantly decreased the endothelial permeability measuredby myocardial absorbance of extravasated Evans Blue dye, compared toIgG-control allografts at 30 min (p<0.01; FIG. 3A). Furthermore,anti-Ang2 perfused allografts had significantly increased microvascularperfusion (P<0.01; FIG. 3B-D).

Doppler velocimeter recording for DA donor rat hearts recovered andintracoronarily administered either anti-Ang2 antibody (MEDI1/5) or withcontrol-IgG, subjected to 4-h cold ischemia and transplanted to fullyMHC-mismatched WF recipient rats were analyzed. The results demonstratedthat the tissue perfusion in the apical myocardium was significantlybetter in the anti-Ang2-treated allografts when compared to theIgG-treated allografts during the first 10 minutes of reperfusion (FIG.3E).

These results show that treatment with a single dose of anti-Ang2antibody to inhibit endogenous Ang2 reduced vascular permeability andincreased the number of perfused myocardial capillaries immediatelyafter reperfusion of the allograft compared to PBS-perfused allografts.

Example 3 Intracoronary Perfusion with Recombinant Ang2 AggravatesIRI-Induced Myocardial Damage

The effect of exogenous, recombinant Ang2 on acute myocardial injury inrat cardiac allograft model was also analyzed. The allografts wereintracoronarily perfused with recombinant Ang2 or control PBS andsubjected to a 4-h hypothermic preservation, transplanted and recoveredat 6-h after reperfusion. Vascular permeability was measured byquantifying myocardial absorbance of extravasated Evans Blue dye withspectrofotometry at 610 nm 30 min after reperfusion. Myocardialcapillary perfusion was measured by quantifying the density ofendothelium-binding FITC-conjugated Lycopersicon esculentum lectin.Vascular permeability and perfusion 30 min after reperfusion remainedunchanged between the groups as shown in FIG. 4A-D. Six (6) h afterreperfusion, intracoronary perfusion with rAng2 increased serum TnT. Theserum levels of cardiomyocyte specific troponin T (TnT) (n=5 to 6 pergroup, P<0.05) is shown in FIG. 4E. These results show that ex vivointracoronary perfusion with exogenous recombinant Ang2 in the rat IRImodel had no effect on permeability and perfusion 30 min afterreperfusion, but increased serum TnT levels, as a sign of extensivemyocardial injury, 6 h after reperfusion compared to PBS. These resultssupport that rAng2 is detrimental to the allograft.

Ex vivo intracoronary perfusion with exogenous recombinant Ang2 alonewas shown to worsen allograft inflammation. The allograft-infiltratingED1+ macrophages, MPO+ neutrophils, CD4+ T cells, CD8+ T cells, andOX62+ dendritic cells 6 h after reperfusion (n=6 per group) are shown inFIG. 4F-J. rAng2 significantly increased the numbers of allograftinfiltrating ED1+ macrophages and MPO+ neutrophils 6 h after reperfusion(which correlated with greater myocardial injury in these groups). Theex vivo intracoronary treatment of allografts with recombinant Ang2(rAng2) alone did not affect microvascular leakage or no-reflowphenomenon 30 min after reperfusion, but rAng2 treatment induced severemyocardial injury and increased inflammatory cell influx when comparedto the PBS-treatment. These results show a pro-inflammatory role forAng2 in IRI in cardiac allografts.

Example 4 Ex Vivo Intracoronary Perfusion with Anti-Ang2 DecreasesIRI-Induced Myocardial Damage and Inflammation in Cardiac Allografts

Cardiomyocytes are highly susceptible to ischemic injury (42). Theeffect of anti-Ang2 antibody (MEDI1/5) on IRI-triggered cardiac damageand on inflammatory responses in transplanted hearts was investigated.

Expression of endothelial Ang2 was measured by quantifying the densityof Ang2/rat endothelial cell antigen (RECA-1) double positive vessels 6h after transplantation (n=6 per group) (FIG. 5A). Myocardial Ang2expression was visualized by immunofluorescence staining for Ang2 andRECA-1 (FIG. 5B). The Mann-Whitney U test was used to compare anti-Ang2and IgG treatment (Scale bars=20 μm). These results showed thatintracoronary perfusion with anti-Ang2 reduced Ang2 expression in theendothelium of the allografts 6 hours after transplantation compared toIgG perfusion (P<0.01).

The allografts were intracoronarily perfused with anti-Ang2 antibody orcontrol IgG and subjected to a 4-h hypothermic preservation,transplanted and recovered at 6-h after reperfusion. Serum levels ofcardiomyocyte specific troponin T (TnT) was measured. In addition,intragraft ED1+ macrophages, MPO+ neutrophils, CD4+ and CD8+ lymphocytesand OX62+DCs were measured. VCAM-1+ vessels were quantified fromhistological images of vessels from control IgG and anti-Ang2 antibodytreated groups, respectively.

Cardiomyocytes are highly susceptible to ischemic injury (Gottlieb R A.J Cardiovasc Pharmacol Ther. 2011; 16(3-4):233-238). The degree ofallograft myocardial injury was assessed by analyzing serum TnT levels.The anti-Ang2 treatment significantly reduced IRI-induced serum TnTrelease compared to control IgG treatment at 6 h (p<0.001; FIG. 6A).Preliminary results showed that using 0.1-fold amount of anti-Ang2antibody (MEDI1/5) did not reduce serum TnT levels, and that using10-fold amount of anti-Ang2 antibody did not provide additional serumTnT release.

Endothelial activation was measured at 6 h after reperfusion, and exvivo intracoronary perfusion with MEDI1/5 significantly reduced vascularcell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1(ICAM-1) immunoreactivity in the microvasculature when compared to IgGtreatment (P<0.01; FIG. 6G-L). The number of graft infiltrating ED1+macrophages, MPO+ neutrophils and CD8+ lymphocytes were alsosignificantly reduced in anti-Ang2 treated allografts compared tocontrol IgG group at 6 h (FIGS. 6B-C and E). The anti-Ang2 antibodytreatment had no significant effect on infiltrating CD4+ lymphocytes orOX62+ dendritic cells (FIGS. 6D and F).

IRI releases target tissue derived endogenous danger molecules. Thesedanger/damage-associated molecular patterns ligate to Toll-likereceptors (TLRs) and form a link between the innate immunity and theactivation of the adaptive immunity that may ultimately be detrimentalfor an allograft. The mRNA expression levels of endogenous TLR-receptorsand ligands 6 h after reperfusion with anti-Ang2 antibody was measured.Compared to IgG treatment, the anti-Ang2 antibody (MEDI1/5) treatmentsignificantly decreased mRNAs encoding innate immune mediators HAS1-3and NFkB, inflammatory cytokines IL-1β, IL-2 and IL-12p35 andtransforming growth factor (TGF)β compared to PBS treatment (Table 2,P<0.05). VEGF increases vascular permeability by directly disruptingEC-EC tight junctions (43), but also by increasing Ang2 production (1).A significant decrease in VEGF mRNA expression in the anti-Ang2 antibodytreated allografts at 6 h after reperfusion was observed compared to PBStreatment (Table 2, P<0.01).

TABLE 2 Gene Anti-Ang2/ctrl-PBS ± SEM p HAS1 0.50 ± 0.07 0.021* HAS20.62 ± 0.10 0.028* HAS3 0.25 ± 0.11 0.010** Biglycan 0.71 ± 0.17 0.105HMGB1 0.78 ± 0.14 0.161 TLR2 0.54 ± 0.10 0.281 TLR4 0.66 ± 0.08 0.779NFkB 0.76 ± 0.08 0.029* IL1b 0.43 ± 0.05 0.040* IL2 0.56 ± 0.15 0.050*IL4 0.70 ± 0.14 0.105 IL6 0.40 ± 0.04 0.073 IL10 0.63 ± 0.14 0.161IL12p35 0.54 ± 0.08 0.007** TNFa 0.35 ± 0.11 0.054 IFNg 0.74 ± 0.210.382 IP10 0.77 ± 0.19 0.328 VEGF-A 0.61 ± 0.05 0.021* CD80 0.66 ± 0.080.442 CD83 0.86 ± 0.11 0.959 CD86 0.81 ± 0.08 0.574 TGFb 0.77 ± 0.060.050* KLF2 0.80 ± 0.07 1.000 Ang1 0.78 ± 0.16 0.161 Ang2 0.87 ± 0.150.721

These results show that the anti-Ang2 antibody treatment efficientlyreduced the immunoreactivity of VCAM-1. Furthermore, these experimentsshowed that the anti-Ang2 antibody treatment significantly decreased theinflux of not only graft infiltrating ED1+ macrophages and MPO+neutrophils, but also CD8+ T cells 6 h after reperfusion.

A decrease in mRNA expression of innate immune activation markers inanti-Ang2 antibody treated allografts 6 h after reperfusion was alsoobserved. Anti-Ang2 antibody treated allografts had significantlyreduced mRNA expression of VEGF and profibrotic TGFβ.

These results show that anti-Ang2 antibody treatment decreases theactivation of endothelium, accumulation of inflammatory cells, andsubsequent acute myocardial injury and innate immune responses.

Example 5 Ex Vivo Intracoronary Perfusion with Anti-Ang2 Ab ReducesCardiac Fibrosis and Allograft Vasculopathy in a Chronic Rejection Model

The effects of ex vivo intracoronary perfusion with anti-Ang2 on cardiacfibrosis and allograft vasculopathy in a chronic rejection model wasanalyzed. Cardiac allografts were perfused with the anti-Ang2 antibody(MEDI1/5) or control IgG and subjected to 2-h preservation to analyzethe effects of anti-Ang2 on cardiac fibrosis and allograft vasculopathyin a chronic rejection model with suboptimal cyclosporine Aimmunosuppression. Under suboptimal immunosuppression with CyA for 8weeks after transplantation, the control allografts developed moderatecardiac fibrosis, which was significantly inhibited by perioperativeintracoronary anti-Ang2 (MEDI1/5) treatment (FIG. 7A-C). In line withthese findings, the Anti-Ang2 (MEDI1/5) treated allografts also hadreduced arteriosclerosis compared to IgG-treated allografts (FIG. 7D-F).When analyzed 8 weeks after transplantation and compared to IgG-treatedallografts, the perioperative ex vivo intracoronary Anti-Ang2 (MEDI1/5)treatment resulted in higher allograft capillary density, measured byquantifying myocardial RECA-1+ vessels (FIG. 7G-I).

These findings also emphasize the importance of early allograftprotection. In a previous study with rat cardiac transplantations, CAVwas shown to be more severe, if the allografts were subjected topreoperative hypothermic (cold) preservation (data not shown). Allograftischemic time is also a known risk factor for mortality in human cardiactransplantation patients. The current results demonstrate thatpreoperative intracoronarily delivered single dose treatment withanti-Ang2 antibody significantly inhibited the development of cardiacfibrosis and vascular occlusion, observed 8 weeks after transplantation.These results show that inhibiting early microvascular dysfunction andIRI results in long-term protection of functional capillary network inrat cardiac allografts.

The current results demonstrate that perioperative treatment with anantibody against endogenous Ang2 prevented microvascular dysfunction andthe development of cardiac fibrosis and allograft vasculpathy. Theseresults support that inhibiting early microvascular dysfunction and IRIwith anti-Ang2 treatment may have long-term effects in cardiacallografts.

Example 6 Human Organ Donor Brain Death and Cardiac TransplantationCauses Changes in Serum Ang2 Levels

Human cardiac allograft donor and recipient blood samples were analyzedfor serum Ang2 and Ang1. Organ donor brain death induces potentiallycardiotoxic systemic cytokine storm (Venkateswaran R V, et al.,Transplantation. 2009; 88:582-588). Samples from eleven cardiacallograft donors and recipients operated between 2010 and 2011 inHelsinki University Central Hospital, Helsinki, Finland were collected.The mean age of the recipients (8 men) was 47.4±11.4 years (range 29-63years). The mean age of the donors (8 men) was 40.9±14.1 years (range19-56 years). Healthy volunteers were used as a control group (n=24).The Ethical Committee of University of Helsinki, Helsinki, Finlandapproved the use of the patient samples.

The serum levels of Ang2 or Ang1 were assessed in human cardiacallograft donors. Blood samples were collected preoperatively fromcardiac allograft donors. Patient serum samples from cardiac allograftdonors (n=11) were analyzed for Ang2 and Ang1 by enzyme-linkedimmunosorbent assay (ELISA) and compared to healthy controls (n=24). TheMann-Whitney U test was used to compare the control group and the donorgroup (FIG. 8A-B).

Patient serum samples from cardiac allograft recipients (n=11) 1, 6, 12,and 24 h after transplantation were analyzed for Ang2 and Ang1 by ELISAand compared to healthy controls (n=24). The Kruskall-Wallis test withDunn correction was used to compare the control group with the recipientgroups. Data were given by box plots showing the upper extreme(excluding outliers), upper quartile, median, lower quartile, and lowerextreme (excluding outliers) (FIG. 8C-D).

ELISA analysis of the serum samples showed that the donor serum Ang2levels were significantly higher than in the control group (P<0.001;FIG. 8A). The serum levels of Ang1 remained unchanged (FIG. 8B). Theeffects of cardiac transplantation on the serum levels of Ang2 and Ang1were also tested. ELISA analysis showed that compared to the controlgroup, Ang2 levels were significantly higher 1, 12 and 24 h aftertransplantation (P<0.05, P<0.001; FIG. 8C). The serum Ang1 levels weresignificantly lower in the recipients 24 h after transplantation(P<0.001; FIG. 8D).

Both the donors and the recipients had higher serum Ang2 levels thanhealthy controls. The Ang2 levels of allograft recipients increasedgradually for up to 24 h after transplantation. These results show thathuman cardiac allograft donors had increased serum Ang2 levels and thatcardiac transplantation resulted in increased serum Ang2 levels up to 24h after transplantation. This Ang2 release in the donor after braindeath can predispose the donated organs to injury and edema, even beforeorgan recovery, hypothermic ischemic preservation, or reperfusion.

Ischemia-reperfusion injury after cardiac transplantation was also shownto increase the serum levels of Ang2 in rats. In particular, thekinetics of early Ang2 release was investigated in rat cardiacallografts. This assay was performed to help determine whether the Ang2release seen in patient samples likely resulted from theischemia-reperfusion injury, or from the alloimmune response. Thekinetics of Ang2 release in rat was analyzed following fully MHCmismatched cardiac transplantations between DA donor and WF recipientrats without immunosuppression. Syngeneic and allogeneic cardiactransplantations were performed wherein DA donor rat hearts wererecovered and subjected to 4-h cold preservation and then transplantedto either DA recipient rats, or to fully MHC-mismatched WF recipientrats without immunosuppression. Serum samples were repeatedly collectedbefore, and 1, 6, and 24 h after cardiac transplantation. Repeatedmeasures ANOVA with the Bonferroni correction revealed that serum levelsof Ang2 gradually rose after reperfusion, peaking 24 h aftertransplantation (FIG. 8E). Similar to the human the patient samples,repeated measures ANOVA revealed significantly increased Ang2 serumlevels of Ang2 in the recipients of rat cardiac allografts 6 and 24 hafter transplantation. However, no Ang2 was detected in serum therecipients of syngenic cardiac allografts. These results suggested thatthe acute alloimmune response, and rather than the IRI, was the keyinducer of Ang2 release after heart transplantation.

Example 7 Hypoxia-Mediated Lung Injury

Anti-Ang2 antibody (MEDI1/5) will be tested to determine if it caninhibit hypoxia-induced lung inflammation and injury. Reports suggeststhat hypoxia induces vascular leakage and inflammation (Eltzschig, H. K.& Carmeliet, P. Hypoxia and inflammation. N Engl J Med 364, 656-665(2011); Eltzschig, H. K. & Eckle, T. Ischemia and reperfusion-frommechanism to translation. Nat Med 17, 1391-1401 (2011)). Angiopoietin-2(Ang2), is considered as a vessel destabilizer and involved ininflammation and destabilization of cell-cell junctions (Augustin, H.G., Koh, G. Y., Thurston, G. & Alitalo, K. Control of vascularmorphogenesis and homeostasis through the angiopoietin-Tie system. NatRev Mol Cell Biol 10, 165-177 (2009)).

Mice receiving ip injection of Ang2 blocking antibody or a controlantibody and iv injection of Evan's Blue will be subjected to hypoxia ina hypoxia chamber supplied with 8% oxygen and 92% nitrogen. The lungswill be harvested and analyzed for Evan's Blue content. In an additionalexperiment, the lungs will be weighed and dried by Speed-Vac. Thewet/dry weight ratio will be used to evaluate pulmonary edema.

Lungs will also be prepared for electron microscopy and histology, e.g.,to see if the vessel junctions are stabilized by Ang2 blockade.

Lungs will be lysed and prepared for reverse-transcription PCR orWestern blot analysis to determine whether Ang2 is upregulated and ifjunctional markers are downregulated during hypoxia, and whether Ang2blocking antibody ameliorates the loss of junctional complexes. Lunglysates will also be subjected to proteomic analysis to detectdifferential signaling between Ang2 inhibition and control duringhypoxia.

In converse experiments, transgenic mice overexpressing Ang2 byendothelial cells in an inducible manner will be analyzed similarly asabove.

Example 8 Ischemic Stroke Injury

Anti-Ang2 antibody (MEDI1/5) will be tested to determine if it caninhibit and/or treat ischemic stroke injury in an animal model forstroke. Reports show that Ang-2 mediates differentiation and migrationof neural progenitor cells in the subventricular zone after stroke (Liuet al., JBC 284(34):22680-22689 (2009)).

Mice receiving ip injection of Ang2 blocking antibody or a controlantibody before or after stroke injury will be subjected to the methodsfor modeling stroke as disclosed in Engel et al., Modeling Stroke inMice—Middle Cerebral Artery Occlusion with the Filament Model. J. Vis.Exp. (47), e2423, DOI: 10.3791/2423 (2011).

In converse experiments, transgenic mice overexpressing Ang2 byendothelial cells in an inducible manner will be analyzed similarly asabove.

Example 9 Ex Vivo Intracoronary Treatment of Rat Cardiac Allografts withAnti-Ang2 Antibody Inhibits Acute and Chronic Rejection

To study the effect of ex vivo intracoronary treatment with anti-Ang2antibody (MEDI1/5) on acute rejection, transplantations in rats wereperformed as described above after 4-h cold preservation, but therecipient rats received a subtherapeutic dose of cyclosporine Aimmunosuppression (2 mg/kg/d for 7 days, and 1 mg/kg/d thereafter). Theresults are shown in FIGS. 9A-N. At 10 d—i.e. at the time of acutealloimmune activation—ex vivo anti-Ang2 antibody treatment significantlyreduced the ICAM-1 immunoreactivity (FIG. 9B) and the numbers ofallograft infiltrating MPO+ neutrophils, CD4+ and CD8+ T cells (FIG.9E-G). In addition, ex vivo intracoronary treatment with anti-Ang2antibody reduced the mRNA expression of Ang2, IL-6, IFN-γ, ICAM-1, CCL3,and TGFβ (FIG. 9I-N).

The effect of ex vivo intracoronary treatment with anti-Ang2 antibody(MEDI1/5) on cardiac fibrosis and allograft vasculopathy in a chronicrejection model was also analyzed. Because previous studies haveindicated that 4-h preoperative cold preservation results inirreversible acute rejection and poor long-term allograft survival, 2-hcold preservation and a subtherapeutic dose of cyclosporine Aimmunosuppression was used in the chronic rejection analysis for thisExample (Tuuminen R, Syrjälä et al., Donor Simvastatin TreatmentAbolishes Rat Cardiac Allograft Ischemia/Reperfusion Injury and ChronicRejection Through Microvascular Protection. Circulation. 2011). In thismodel, the allografts in both groups survived about equally as long(FIG. 90) and were compared for histological changes (see FIG. 7A-Iabove).

These results show that Ang-2 had a prominent pro-inflammatory roleimmediately after transplantation and that inhibiting this response withanti-Ang2 antibody (MEDI1/5) resulted in long-term protection of cardiacallografts.

Example 10 Recipient Treatment with Single Dose of Anti-Ang2 AntibodyInhibits Ischemia-Reperfusion Injury by Anti-Inflammatory Function inRat Cardiac Allografts

Donor rat hearts were subjected to 4-h cold ischemia and the recipientsreceived anti-Ang2 antibody (MEDI1/5) or control-IgG (1 mg/kg) i.p. 4 hbefore the transplantation. Modified Miles assay showed that theanti-Ang2 antibody did not protect the allograft from microvascularleakage and no-reflow phenomenon 30 min after the reperfusion (FIG.10A-D).

The serum levels of Ang2 increased during the first day after theallograft transplantation, but not after syngraft transplantation, whichshows that systemic recipient treatment with anti-Ang2 antibody wouldlikely have an impact on early allograft inflammation. The allograftsfrom DA donors were subjected to 4-h cold ischemia, and the WFrecipients received anti-Ang2 antibody (MEDI1/5) or control-IgG (1mg/kg) i.p. 4 h before the transplantation. This reduced the serumlevels of TnT, the immunoreactivity of VCAM-1 and ICAM-1, and the numberof allograft-infiltrating ED1+ macrophages, MPO+ neutrophils, and CD8+ Tcells 6 h after the reperfusion, when compared to IgG-treated recipients(FIG. 10E-L).

Example 11 Recipient Treatment With Multiple Doses of Anti-Ang2 AntibodyProlongs the Survival of Rat Cardiac Allografts

The effect of single and multiple anti-Ang2 antibody doses on allograftsurvival were compared to further explore the anti-inflammatory effectsof recipient anti-Ang2 antibody treatment. The recipients received avery low dose of cyclosporine A (1 mg/kg/d s.c.) and either a singlepreoperative i.p. dose of anti-Ang2 antibody (MEDI1/5) or IgG, or 4consecutive doses of anti-Ang2 antibody (MEDI1/5) or IgG 4 hour beforetransplantation, and 1, 3, and 5 days after the transplantation.Recipient treatment with single dose of anti-Ang2 antibody did notprolong cardiac allograft survival compared to the IgG-treatedrecipients (20.5±18.1 vs 10.2±2.3 days; FIG. 11A). However, multipledoses of anti-Ang2 antibody significantly prolonged allograft survivalcompared to the IgG-treated recipients (30.5±20.3 vs 9.6±1.5 days; FIG.11B). These results show that Ang2 regulates inflammatory responses incardiac allografts, in particular during the first days aftertransplantation, and administration of multiple doses of anti-Ang2antibody to transplant recipients was able to prolonged allograftsurvival.

Although systemic treatment of allograft recipients with anti-Ang2antibody 4 h before transplantation did not prevent microvascularleakage and the no-reflow phenomenon, the treatment significantlyreduced allograft myocardial injury, microvascular endothelial cellactivation, and inflammatory cell influx. Furthermore, when threeconsecutive doses of anti-Ang2 antibody were given during the following5 days after transplantation, the allograft survival was prolonged andthe development of cardiac fibrosis and allograft vasculopathy wasprevented. Thus, in systemic recipient treatment, a multiple doseregimen of anti-angiopoietin-2 antibody produced beneficial long-termeffects.

Example 12 Recipient Treatment with Multiple Doses of Anti-Ang2 AntibodyPrevents Acute and Chronic Rejection in Rat Cardiac Allografts

Multiple doses of anti-Ang2 antibody were tested to determine if thistreatment modality could prevent acute rejection and the development ofcardiac fibrosis and allograft vasculopathy in the chronic rejectionmodel in rats. To assess the effect of recipient treatment on thealloimmune responses, the recipients received 4 consecutive i.p. dosesof anti-Ang2 antibody (MEDI1/5) as above, plus subclinicalimmunosuppression with daily cyclosporine A (2 mg/kg/d for 7 days, and 1mg/kg/d thereafter). Ten (10) days after the reperfusion, the recipienttreatment with multiple doses of anti-Ang2 antibody significantlyreduced the density of ICAM1+ capillaries, and the numbers of allograftinfiltrating ED1+ macrophages, CD4+ and CD8+ T cells, and OX62+dendritic cells (FIG. 12A-H). In chronic rejection experiments, thesurvival of the allografts was similar in both groups (FIG. 121).Recipient treatment with multiple doses of anti-Ang2 antibodysignificantly increased the myocardial capillary density (FIGS. 12J-L),and prevented the development of cardiac fibrosis and allograftvasculopathy 8 weeks after transplantation (FIGS. 12M-O and 12P-R).

Example 13 Ex Vivo Intracoronary Treatment of Rat Cardiac Allograftswith Anti-Ang2 Antibody Prevents Ischemia-Reperfusion Injury Via anAnti-Inflammatory Function

The degree of allograft myocardial injury was assessed by measuringserum levels of cardiac troponin T (TnT) in serum 6 h after thetransplantation. First, a dose-response analysis was performed, in whichthe ex vivo intracoronary treatment with anti-Ang2 antibody (MEDI1/5)with the dose of 3 μg/g (0.1×) of heart weight failed to inhibit acutemyocardial injury, but the doses of 30 μg/g (lx) and 300 μg/g (10×) ofheart weight reduced the serum levels of TnT significantly compared toIgG-treatment (FIG. 13A). Based on these results, the dose of 30 μg/g ofheart weight was used in subsequent experiments.

To confirm the specificity of the anti-Ang2 antibody (MEDI1/5) inprotection from ischemia-reperfusion injury, add-back experiments wereperformed in which anti-Ang2 antibody (30 ng/g MEDI1/5) in combinationwith rAng2 (2 μg/g) was administered. The results shows that rAng2abrogated the positive result of the ex vivo intracoronary treatmentwith the anti-Ang2 antibody (FIG. 13A). As discussed above, the ex vivointracoronary treatment of allografts with recombinant Ang2 (rAng2)alone did not affect microvascular leakage or no-reflow phenomenon 30min after reperfusion, but rAng2 treatment induced severe myocardialinjury and increased inflammatory cell influx when compared to thePBS-treatment (FIG. 4F-J).

Ex vivo intracoronary treatment of the allografts with the anti-Ang2antibody significantly reduced vascular cell adhesion molecule-1(VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and P-selectinimmunoreactivity in the microvasculature 6 h after the reperfusion, whencompared to the IgG treatment (FIGS. 6G, 6J, and 13B). Furthermore,anti-Ang2 antibody treatment reduced the numbers of allograftinfiltrating ED1+ macrophages, MPO+ neutrophils, and CD8+ T cells (FIGS.13E, D, and F). In contrast, the numbers of allograft infiltrating CD4+T cells or OX62+ dendritic cells were not changed (FIGS. 6E and G).These results show that anti-Ang2 antibody (MEDI1/5) treatment preventedthe microvascular endothelial cell activation, accumulation ofinflammatory cells, and innate immune activation in the allograft.

Ex vivo intracoronary treatment with rAng2 or—in add-backexperiments—with rAng2 and anti-Ang2 antibody significantly increasedthe numbers of allograft infiltrating ED1+ macrophages and MPO+neutrophils 6 h after the reperfusion, which correlated with greatermyocardial injury in these groups (FIGS. 4F-J and 14A-G). Addingrecombinant Ang2 to ex vivo intracoronary perfusion solution abrogatedthe protective effects of anti-Ang2 antibody 6 h after reperfusion. Thedensity of VCAM-1+, ICAM-1+ vessels in myocardial cross sections 6 hafter reperfusion are shown in FIG. 14A-B. The numbers of intragraftED1+ macrophages, MPO+ neutrophils, CD4+ T cells, CD8+ T cells, andOX62+ dendritic cells 6 h after reperfusion are shown FIGS. 14C-G.Together, these results suggest that Ang2 had a pro-inflammatory role inIRI in cardiac allografts.

The results above show that early after reperfusion, regardless of thedelivery method, anti-Ang2 antibody treatment significantly decreasedthe influx of graft-infiltrating macrophages, neutrophils, and CD8+ Tcells early after the reperfusion.

III. Discussion

In vitro, hypoxia induced the deposition of Ang2-Tie2 complexes atendothelial cell-cell junctions. Ex vivo intracoronary perfusion with anAng2 blocking antibody prevented destabilization of endothelialcell-cell junctions in coronary microvasculature during allograftpreservation. Furthermore, ex vivo intracoronary perfusion withanti-Ang2 antibody reduced vascular leakage, no-reflow phenomenon,endothelial activation, inflammatory cell influx, myocardial injury, andinnate immune activation in cardiac allografts after reperfusion. In achronic rejection model, ex vivo intracoronary perfusion with anti-Ang2antibody reduced adaptive immune response, cardiac fibrosis, andallograft vasculopathy and maintained myocardial capillary density.

The effect of intracoronarily delivered anti-Ang2 antibody onIRI-induced microvascular dysfunction and subsequent development ofcardiac fibrosis and allograft vasculopathy in rat cardiac allograftswas investigated. The results using an experimental cardiactransplantation model showed that anti-Ang2 antibody administration wasadvantageous for the protection of rat cardiac allografts from IRI. Byinjecting anti-Ang2 antibody into the coronaries via the clamped aorticroot of a recovered heart, the disruption of vascular permeabilityassociated with IRI and the subsequent myocardial damage was prevented.The results disclosed herein show a clinically feasible method ofallograft protection with an intracoronarily administered single dose ofendogenous Ang2 neutralizing anti-Ang2 antibody. Anti-Ang2 antibodytherapy inhibited the IRI-induced microvascular dysfunction andsubsequent development of cardiac fibrosis and chronic allograftvasculopathy. Single dose treatment with anti-Ang2 inhibitedIRI-associated early innate immune activation by multiple parallelmechanisms and, furthermore, inhibited the development of subsequentcardiac fibrosis and CAV. These results show a clinically feasiblestrategy to protect cardiac allografts from primary and long-termallograft dysfunction, e.g., anti-Ang2 antibody use for clinical use inprimary allograft protection as well as in perfusion solutionsmaintaining the endothelial integrity during the transplantationsurgery.

These results show that Ang2 has a critical role in rat cardiacallograft IRI and microvascular dysfunction. Intracoronary perfusionwith anti-Ang2 antibodies resulted in both primary and long-termallograft function improvement and protection from allograftdysfunction. These results support targeting the Ang1/Ang2 axis withanti-Ang2 antibody in protection of solid organ transplants.Furthermore, the results show preoperative ex vivo intracoronaryperfusion with the anti-Ang2 antibody decreased IRI-associated vascularleakage, no-reflow phenomenon, cardiomyocyte damage, innate immuneactivation and subsequent development of cardiac fibrosis and allograftvasculopathy. These results show an important role for Ang2 in IRI andactivation of innate and adaptive immune responses in cardiac allograftsand support a clinically feasible, single ex vivo injection strategy toprotect cardiac allografts from primary and long-term allograftdysfunction. The results highlight the role of Ang2 in regulatingIRI-induced vascular dysfunction and support the use of anti-Ang2antibody as a therapy for solid organ transplant protection.

In cardiac syngrafts, the primary IRI-induced neutrophil-intensiveinflammation subsides during the first 24 h after heart thetransplantation. However, in the cardiac allografts, the presence ofCD8+ T cells prolongs the inflammatory response of neutrophils for up tothree days after heart the transplantation. The results above show thatearly after reperfusion, regardless of the delivery method, anti-Ang2antibody treatment significantly decreased the influx ofgraft-infiltrating macrophages, neutrophils, and, CD8+ T cells. Theseresults further support a T cell-independent strategy to protect cardiacallografts with long-term beneficial effects.

The results herein show that the brain-death and the alloimmune responseafter transplantation induced the release of Ang2, and that ex vivointracoronary treatment of rat cardiac allografts with anti-Ang2blocking antibody prevented microvascular leakage and the no-reflowphenomenon. Furthermore, ex vivo intracoronary treatment of allograftsand systemic recipient treatment with anti-Ang2 antibody inhibited theearly innate and adaptive immune responses and the development ofcardiac fibrosis and allograft vasculopathy. These results demonstratethe pivotal role of Ang2 in innate and adaptive immune responses afterheart transplantation, and provide a clinically feasible strategy toprevent microvascular injury and the activation of innate immunity, andto protect cardiac allografts from the development of primary andlong-term allograft failure using anti-Ang2 blocking antibody.

The present invention has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternate boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

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What is claimed is: 1.-5. (canceled)
 6. A method for protecting a solidorgan transplant tissue, comprising administering an effective amount ofan anti-Ang2 antibody or antigen-binding fragment thereof to anallograft. 7.-11. (canceled)
 12. A method for treating or amelioratingmyocardial ischemia, comprising administering a therapeuticallyeffective amount of an anti-Ang2 antibody or antigen-binding fragmentthereof to a subject in need thereof.
 13. A method for treating anIRI-induced inflammatory response in a subject comprising administeringa therapeutically effective amount of an anti-Ang2 antibody orantigen-binding fragment thereof to the subject in need thereof. 14.(canceled)
 15. The method of claim 6, wherein the anti-Ang2 antibody orantigen-binding fragment thereof neutralizes Ang-2.
 16. The method ofclaim 6, wherein the anti-Ang2 antibody or antigen-binding fragmentthereof preferentially binds Ang-2 over Ang-1.
 17. The method of claim6, wherein the anti-Ang2 antibody or antigen-binding fragment thereof isan antibody that binds to and neutralizes Ang-2, but does not bind toAng-1. 18.-37. (canceled)
 38. The method of claim 12, wherein theanti-Ang2 antibody or antigen-binding fragment thereof neutralizesAng-2.
 39. The method of claim 12, wherein the anti-Ang2 antibody orantigen-binding fragment thereof preferentially binds Ang-2 over Ang-1.40. The method of claim 12, wherein the anti-Ang2 antibody orantigen-binding fragment thereof is an antibody that binds to andneutralizes Ang-2, but does not bind to Ang-1.
 41. The method of claim13, wherein the anti-Ang2 antibody or antigen-binding fragment thereofneutralizes Ang-2.
 42. The method of claim 13, wherein the anti-Ang2antibody or antigen-binding fragment thereof preferentially binds Ang-2over Ang-1.
 43. The method of claim 13, wherein the anti-Ang2 antibodyor antigen-binding fragment thereof is an antibody that binds to andneutralizes Ang-2, but does not bind to Ang-1.